Influenza virus vaccines and uses thereof

ABSTRACT

Provided herein are chimeric influenza hemagglutinin (HA) polypeptides, compositions comprising the same, vaccines comprising the same, and methods of their use.

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/738,672, filed Dec. 18, 2012, and U.S. Provisional PatentApplication No. 61/840,899, filed Jun. 28, 2013, the disclosure of eachof which is incorporated herein by reference in its entirety.

This invention was made with government support under Grant Nos.AI070469, AI086061 and HHSN266200700010C awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

1. INTRODUCTION

Provided herein are chimeric influenza virus hemagglutinin polypeptidesand compositions comprising the same, vaccines comprising the same, andmethods of their use.

2. BACKGROUND

Influenza viruses are enveloped RNA viruses that belong to the family ofOrthomyxoviridae (Palese and Shaw (2007) Orthomyxoviridae: The Virusesand Their Replication, 5th ed. Fields' Virology, edited by B. N. Fields,D. M. Knipe and P. M. Howley. Wolters Kluwer Health/Lippincott Williams& Wilkins, Philadelphia, USA, p 1647-1689). The natural host ofinfluenza A viruses are mainly avians, but influenza A viruses(including those of avian origin) also can infect and cause illness inhumans and other animal hosts (bats, canines, pigs, horses, sea mammals,and mustelids). For example, the H5N1 avian influenza A viruscirculating in Asia has been found in pigs in China and Indonesia andhas also expanded its host range to include cats, leopards, and tigers,which generally have not been considered susceptible to influenza A(CIDRAP—Avian Influenza: Agricultural and Wildlife Considerations). Theoccurrence of influenza virus infections in animals could potentiallygive rise to human pandemic influenza strains.

Influenza A and B viruses are major human pathogens, causing arespiratory disease that ranges in severity from sub-clinical infectionto primary viral pneumonia which can result in death. The clinicaleffects of infection vary with the virulence of the influenza strain andthe exposure, history, age, and immune status of the host. Thecumulative morbidity and mortality caused by seasonal influenza issubstantial due to the relatively high attack rate. In a normal season,influenza can cause between 3-5 million cases of severe illness and upto 500,000 deaths worldwide (World Health Organization (2003) Influenza:Overview; March 2003). In the United States, influenza viruses infect anestimated 10-15% of the population (Glezen and Couch R B (1978)Interpandemic influenza in the Houston area, 1974-76. N Engl J Med 298:587-592; Fox et al. (1982) Influenza virus infections in Seattlefamilies, 1975-1979. II. Pattern of infection in invaded households andrelation of age and prior antibody to occurrence of infection andrelated illness. Am J Epidemiol 116: 228-242) and are associated withapproximately 30,000 deaths each year (Thompson W W et al. (2003)Mortality Associated with Influenza and Respiratory Syncytial Virus inthe United States. JAMA 289: 179-186; Belshe (2007) Translationalresearch on vaccines: influenza as an example. Clin Pharmacol Ther 82:745-749).

In addition to annual epidemics, influenza viruses are the cause ofinfrequent pandemics. For example, influenza A viruses can causepandemics such as those that occurred in 1918, 1957, 1968, and 2009. Dueto the lack of pre-formed immunity against the major viral antigen,hemagglutinin (HA), pandemic influenza can affect greater than 50% ofthe population in a single year and often causes more severe diseasethan epidemic influenza. A stark example is the pandemic of 1918, inwhich an estimated 50-100 million people were killed (Johnson andMueller (2002) Updating the Accounts: Global Mortality of the 1918-1920“Spanish” Influenza Pandemic Bulletin of the History of Medicine 76:105-115). Since the emergence of the highly pathogenic avian H5N1influenza virus in the late 1990s (Claas et al. (1998) Human influenza AH5N1 virus related to a highly pathogenic avian influenza virus. Lancet351: 472-7), there have been concerns that it may be the next pandemicvirus. Further, H7 and H9 strains are candidates for new pandemics sincethese strains infect humans on occasion.

An effective way to protect against influenza virus infection is throughvaccination; however, current vaccination approaches rely on achieving agood match between circulating strains and the isolates included in thevaccine. Such a match is often difficult to attain due to a combinationof factors. First, influenza viruses are constantly undergoing change:every 3-5 years the predominant strain of influenza A virus is replacedby a variant that has undergone sufficient antigenic drift to evadeexisting antibody responses. Isolates to be included in vaccinepreparations must therefore be selected each year based on the intensivesurveillance efforts of the World Health Organization (WHO)collaborating centers. Second, to allow sufficient time for vaccinemanufacture and distribution, strains must be selected approximately sixmonths prior to the initiation of the influenza season. Often, thepredictions of the vaccine strain selection committee are inaccurate,resulting in a substantial drop in the efficacy of vaccination.

The possibility of a novel subtype of influenza A virus entering thehuman population also presents a significant challenge to currentvaccination strategies. Since it is impossible to predict what subtypeand strain of influenza virus will cause the next pandemic, current,strain-specific approaches cannot be used to prepare a pandemicinfluenza vaccine.

3. SUMMARY

In one aspect, provided herein are chimeric influenza hemagglutinin (HA)polypeptides that induce a cross-protective immune response against theconserved HA stem domain of influenza viruses. The chimeric influenza HApolypeptides provided herein comprise a stable (e.g., properly formed)HA stem domain and a globular HA head domain that is heterologous to thestem domain (i.e. the head and stem domains are derived from differentstrains and/or subtypes of influenza virus).

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza virus of the H1 subtype and (ii) theglobular head domain of the hemagglutinin from an influenza virus of theH5 subtype (sometimes referred to herein as a “cH5/1 chimeric influenzahemagglutinin polypeptide”). In a specific embodiment, the stem domainof a cH5/1 chimeric influenza hemagglutinin polypeptide is the stemdomain of A/California/4/2009 (H1N1) HA (or the stem domain of anA/California/4/2009-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/1 chimeric influenza hemagglutininpolypeptide is the stem domain of A/California/4/2009 (H1N1) HA (or thestem domain of an A/California/4/2009-like influenza virus HA) and theglobular head domain of the cH5/1 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Vietnam/1203/2004 (H5) HA(or the globular head domain of an A/Vietnam/1203/2004 (H5)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH5/1 chimeric influenza hemagglutinin polypeptide is the stem domainof A/California/4/2009 (H1N1) HA (or the stem domain of anA/California/4/2009-like influenza virus HA) and the globular headdomain of the cH5/1 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Indonesia/5/2005 (H5) HA (or the globular headdomain of an A/Indonesia/5/2005 (H5)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH5/1 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/California/4/2009 (H1N1) HA (or the stem domain of anA/California/4/2009-like influenza virus HA) and the globular headdomain of the cH5/1 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Anhui/1/2005 (H5) HA (or the globular headdomain of an A/Anhui/1/2005 (H5)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH5/1 chimeric influenzahemagglutinin polypeptide is the stem domain of A/California/4/2009(H1N1) HA (or the stem domain of an A/California/4/2009-like influenzavirus HA) and the globular head domain of the cH5/1 chimeric influenzahemagglutinin polypeptide is the globular head domain of A/Bar headedgoose/Quinghai/1A/2005 (H5) HA (or the globular head domain of an A/Barheaded goose/Quinghai/1A/2005 (H5)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH5/1 chimeric influenzahemagglutinin polypeptide is the stem domain of A/California/4/2009(H1N1) HA (or the stem domain of an A/California/4/2009-like influenzavirus HA) and the globular head domain of the cH5/1 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/turkey/Turkey/1/2005 (H5) HA (or the globular head domain of anA/turkey/Turkey/1/2005 (H5)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH5/1 chimeric influenzahemagglutinin polypeptide is the stem domain of A/California/4/2009(H1N1) HA (or the stem domain of an A/California/4/2009-like influenzavirus HA) and the globular head domain of the cH5/1 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Whooperswan/Mongolia/244/2005 (H5) HA (or the globular head domain ofan A/Whooperswan/Mongolia/244/2005 (H5)-like influenza virus HA). Inspecific embodiments, the stem domain of the hemagglutinin from aninfluenza virus of the H1 subtype of a cH5/1 chimeric influenzahemagglutinin polypeptide provided herein is from an H1 subtype that themajority of the population is naive to. In certain embodiments, the stemdomain of the hemagglutinin from an influenza virus of the H1 subtype ofa cH5/1 chimeric influenza hemagglutinin polypeptide provided herein isfrom an upcoming H1N1 vaccine strain, e.g., the H1N1 vaccine strain inuse in the year 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021,2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2032,2033, 2034, or 2035.

In a specific embodiment, a cH5/1 chimeric influenza hemagglutininpolypeptide does not comprise the stem domain of A/Puerto Rico/8/34(“PR8”) HA and does not comprise the globular head domain ofA/Vietnam/1203/2004 (H5) HA.

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza virus of the H3 subtype and (ii) theglobular head domain of the hemagglutinin from an influenza virus of theH5 subtype (sometimes referred to herein as a “cH5/3 chimeric influenzahemagglutinin polypeptide”). In a specific embodiment, the stem domainof a cH5/3 chimeric influenza hemagglutinin polypeptide is the stemdomain of A/Victoria/361/2011 (H3N2) HA (or the stem domain of anA/Victoria/361/2011 (H3N2)-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Victoria/361/2011 (H3N2) HA (or thestem domain of an A/Victoria/361/2011 (H3N2)-like influenza virus HA)and the globular head domain of the cH5/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Vietnam/1203/2004 (H5) HA (or the globular head domain of anA/Vietnam/1203/2004 (H5)-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Victoria/361/2011 (H3N2) (or thestem domain of an A/Victoria/361/2011 (H3N2)-like influenza virus HA) HAand the globular head domain of the cH5/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Indonesia/5/2005 (H5) HA (or the globular head domain of anA/Indonesia/5/2005 (H5)-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Victoria/361/2011 (H3N2) HA (or thestem domain of an A/Victoria/361/2011 (H3N2)-like influenza virus HA)and the globular head domain of the cH5/3 chimeric influenzahemagglutinin polypeptide is the globular head domain of A/Anhui/1/2005(H5) HA (or the globular head domain of an A/Anhui/1/2005 (H5)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH5/3 chimeric influenza hemagglutinin polypeptide is the stem domainof A/Victoria/361/2011 (H3N2) HA (or the stem domain of anA/Victoria/361/2011 (H3N2)-like influenza virus HA) and the globularhead domain of the cH5/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA(or the globular head domain of an A/Bar headed goose/Quinghai/1A/2005(H5)-like influenza virus HA). In another specific embodiment, the stemdomain of a cH5/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Victoria/361/2011 (H3N2) HA (or the stem domain of anA/Victoria/361/2011 (H3N2)-like influenza virus HA) and the globularhead domain of the cH5/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/turkey/Turkey/1/2005 (H5) HA (or theglobular head domain of an A/turkey/Turkey/1/2005 (H5)-like influenzavirus HA). In another specific embodiment, the stem domain of a cH5/3chimeric influenza hemagglutinin polypeptide is the stem domain ofA/Victoria/361/2011 (H3N2) HA (or the stem domain of anA/Victoria/361/2011 (H3N2)-like influenza virus HA) and the globularhead domain of the cH5/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Whooperswan/Mongolia/244/2005 (H5) HA (orthe globular head domain of an A/Whooperswan/Mongolia/244/2005 (H5)-likeinfluenza virus HA).

In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA (or the stem domain of an A/harborseal/Massachusetts/1/2011 (H3N8)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH5/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA (or the stem domain of an A/harborseal/Massachusetts/1/2011 (H3N8)-like influenza virus HA) and theglobular head domain of the cH5/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Vietnam/1203/2004 (H5) HA(or the globular head domain of an A/Vietnam/1203/2004 (H5)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH5/3 chimeric influenza hemagglutinin polypeptide is the stem domainof A/harbor seal/Massachusetts/1/2011 (H3N8) HA (or the stem domain ofan A/harbor seal/Massachusetts/1/2011 (H3N8)-like influenza virus HA)and the globular head domain of the cH5/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Indonesia/5/2005 (H5) HA (or the globular head domain of anA/Indonesia/5/2005 (H5)-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/harbor seal/Massachusetts/1/2011(H3N8) HA (or the stem domain of an A/harbor seal/Massachusetts/1/2011(H3N8)-like influenza virus HA) and the globular head domain of thecH5/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Anhui/1/2005 (H5) HA (or the globular head domain of anA/Anhui/1/2005 (H5)-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/harbor seal/Massachusetts/1/2011(H3N8) HA (or the stem domain of an A/harbor seal/Massachusetts/1/2011(H3N8)-like influenza virus HA) and the globular head domain of thecH5/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA (or the globularhead domain of an A/Bar headed goose/Quinghai/1A/2005 (H5)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH5/3 chimeric influenza hemagglutinin polypeptide is the stem domainof A/harbor seal/Massachusetts/1/2011 (H3N8) (or the stem domain of anA/harbor seal/Massachusetts/1/2011 (H3N8)-like influenza virus HA) HAand the globular head domain of the cH5/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/turkey/Turkey/1/2005 (H5) HA (or the globular head domain of anA/turkey/Turkey/1/2005 (H5)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH5/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA (or the stem domain of an A/harborseal/Massachusetts/1/2011 (H3N8)-like influenza virus HA) and theglobular head domain of the cH5/3 chimeric influenza hemagglutininpolypeptide is the globular head domain ofA/Whooperswan/Mongolia/244/2005 (H5) HA (or the globular head domain ofan A/Whooperswan/Mongolia/244/2005 (H5)-like influenza virus HA).

In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA (or the stem domain of an A/Indiana/10/2011(H3N2)-like influenza virus HA). In another specific embodiment, thestem domain of a cH5/3 chimeric influenza hemagglutinin polypeptide isthe stem domain of A/Indiana/10/2011 (H3N2) HA (or the stem domain of anA/Indiana/10/2011 (H3N2)-like influenza virus HA) and the globular headdomain of the cH5/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Vietnam/1203/2004 (H5) HA (or the globularhead domain of an A/Vietnam/1203/2004 (H5)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA (or the stem domain of an A/Indiana/10/2011(H3N2)-like influenza virus HA) and the globular head domain of thecH5/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Indonesia/5/2005 (H5) HA (or the globular head domain of anA/Indonesia/5/2005 (H5)-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Indiana/10/2011 (H3N2) (or the stemdomain of an A/Indiana/10/2011 (H3N2)-like influenza virus HA) HA andthe globular head domain of the cH5/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Anhui/1/2005 (H5) HA (orthe globular head domain of an A/Anhui/1/2005 (H5)-like influenza virusHA). In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA (or the stem domain of an A/Indiana/10/2011(H3N2)-like influenza virus HA) and the globular head domain of thecH5/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA (or the globularhead domain of an A/Bar headed goose/Quinghai/1A/2005 (H5)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH5/3 chimeric influenza hemagglutinin polypeptide is the stem domainof A/Indiana/10/2011 (H3N2) HA (or the stem domain of anA/Indiana/10/2011 (H3N2)-like influenza virus HA) and the globular headdomain of the cH5/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/turkey/Turkey/1/2005 (H5) HA (or the globularhead domain of an A/turkey/Turkey/1/2005 (H5)-like influenza virus HA).In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA (or the stem domain of an A/Indiana/10/2011(H3N2)-like influenza virus HA) and the globular head domain of thecH5/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Whooperswan/Mongolia/244/2005 (H5) HA (or the globular headdomain of an A/Whooperswan/Mongolia/244/2005 (H5)-like influenza virusHA).

In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA (or the stem domain of an A/Perth/16/2009(H3N2)-like influenza virus HA). In another specific embodiment, thestem domain of a cH5/3 chimeric influenza hemagglutinin polypeptide isthe stem domain of A/Perth/16/2009 (H3N2) HA (or the stem domain of anA/Perth/16/2009 (H3N2)-like influenza virus HA) and the globular headdomain of the cH5/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Vietnam/1203/2004 (H5) HA (or the globularhead domain of an A/Vietnam/1203/2004 (H5)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA (or the stem domain of an A/Perth/16/2009(H3N2)-like influenza virus HA) and the globular head domain of thecH5/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Indonesia/5/2005 (H5) HA (or the globular head domain of anA/Indonesia/5/2005 (H5)-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Perth/16/2009 (H3N2) HA (or the stemdomain of an A/Perth/16/2009 (H3N2)-like influenza virus HA) and theglobular head domain of the cH5/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Anhui/1/2005 (H5) HA (orthe globular head domain of an A/Anhui/1/2005 (H5)-like influenza virusHA). In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA (or the stem domain of an A/Perth/16/2009(H3N2)-like influenza virus HA) and the globular head domain of thecH5/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA (or the globularhead domain of an A/Bar headed goose/Quinghai/1A/2005 (H5)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH5/3 chimeric influenza hemagglutinin polypeptide is the stem domainof A/Perth/16/2009 (H3N2) HA (or the stem domain of an A/Perth/16/2009(H3N2)-like influenza virus HA) and the globular head domain of thecH5/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/turkey/Turkey/1/2005 (H5) HA (or the globular head domain ofan A/turkey/Turkey/1/2005 (H5)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH5/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/Perth/16/2009 (H3N2)HA (or the stem domain of an A/Perth/16/2009 (H3N2)-like influenza virusHA) and the globular head domain of the cH5/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Whooperswan/Mongolia/244/2005 (H5) HA (or the globular head domain ofan A/Whooperswan/Mongolia/244/2005 (H5)-like influenza virus HA).

In specific embodiments, the stem domain of the hemagglutinin from aninfluenza virus of the H3 subtype of a cH5/3 chimeric influenzahemagglutinin polypeptide provided herein is from an H3 subtype that themajority of the population is naive to. In certain embodiments, the stemdomain of the hemagglutinin from an influenza virus of the H3 subtype ofa cH5/3 chimeric influenza hemagglutinin polypeptide provided herein isfrom an upcoming H3N2 vaccine strain, e.g., the H3N2 vaccine strain inuse in the year 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021,2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2032,2033, 2034, or 2035.

In a specific embodiment, a cH5/3 chimeric influenza hemagglutininpolypeptide provided herein does not comprise the globular head domainof A/Vietnam/1203/2004 (H5) HA and does not comprise the stem domain ofA/Perth/16/2009 (H3) HA.

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza virus of the H3 subtype and (ii) theglobular head domain of the hemagglutinin from an influenza virus of theH7 subtype (sometimes referred to herein as a “cH7/3 chimeric influenzahemagglutinin polypeptide”).

In a specific embodiment, the stem domain of a cH7/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/Victoria/361/2011(H3N2) HA (or the stem domain of an A/Victoria/361/2011 (H3N2)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/3 chimeric influenza hemagglutinin polypeptide is the stem domainof A/Victoria/361/2011 (H3N2) HA (or the stem domain of anA/Victoria/361/2011 (H3N2)-like influenza virus HA) and the globularhead domain of the cH7/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Netherlands/219/03 (H7) HA (or theglobular head domain of an A/Netherlands/219/03 (H7)-like influenzavirus HA). In another specific embodiment, the stem domain of a cH7/3chimeric influenza hemagglutinin polypeptide is the stem domain ofA/Victoria/361/2011 (H3N2) HA (or the stem domain of anA/Victoria/361/2011 (H3N2)-like influenza virus HA) and the globularhead domain of the cH7/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Canada/504/04 (H7) HA (or the globularhead domain of an A/Canada/504/04 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Victoria/361/2011 (H3N2) HA (or the stem domain of anA/Victoria/361/2011 (H3N2)-like influenza virus HA) and the globularhead domain of the cH7/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Canada/444/04 (H7) HA (or the globularhead domain of an A/Canada/444/04 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Victoria/361/2011 (H3N2) HA (or the stem domain of anA/Victoria/361/2011 (H3N2)-like influenza virus HA) and the globularhead domain of the cH7/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/chicken/Jalisco/CPA1/2012 (H7) HA (or theglobular head domain of an A/chicken/Jalisco/CPA1/2012 (H7)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/3 chimeric influenza hemagglutinin polypeptide is the stem domainof A/Victoria/361/2011 (H3N2) HA (or the stem domain of anA/Victoria/361/2011 (H3N2)-like influenza virus HA) and the globularhead domain of the cH7/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/mallard/Alberta/24/2001 (H7) HA (or theglobular head domain of an A/mallard/Alberta/24/2001 (H7)-like influenzavirus HA). In another specific embodiment, the stem domain of a cH7/3chimeric influenza hemagglutinin polypeptide is the stem domain ofA/Victoria/361/2011 (H3N2) HA (or the stem domain of anA/Victoria/361/2011 (H3N2)-like influenza virus HA) and the globularhead domain of the cH7/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Rhea/NC/39482/93 (H7) HA (or the globularhead domain of an A/Rhea/NC/39482/93 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Victoria/361/2011 (H3N2) HA (or the stem domain of anA/Victoria/361/2011 (H3N2)-like influenza virus HA) and the globularhead domain of the cH7/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/mallard/Netherlands/12/2000 (H7) HA (orthe globular head domain of an A/mallard/Netherlands/12/2000 (H7)-likeinfluenza virus HA).

In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA (or the stem domain of an A/harborseal/Massachusetts/1/2011 (H3N8)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH7/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA (or the stem domain of an A/harborseal/Massachusetts/1/2011 (H3N8)-like influenza virus HA) and theglobular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Netherlands/219/03 (H7) HA(or the globular head domain of an A/Netherlands/219/03 (H7)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/3 chimeric influenza hemagglutinin polypeptide is the stem domainof A/harbor seal/Massachusetts/1/2011 (H3N8) HA (or the stem domain ofan A/harbor seal/Massachusetts/1/2011 (H3N8)-like influenza virus HA)and the globular head domain of the cH7/3 chimeric influenzahemagglutinin polypeptide is the globular head domain of A/Canada/504/04(H7) HA (or the globular head domain of an A/Canada/504/04 (H7)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/3 chimeric influenza hemagglutinin polypeptide is the stem domainof A/harbor seal/Massachusetts/1/2011 (H3N8) HA (or the stem domain ofan A/harbor seal/Massachusetts/1/2011 (H3N8)-like influenza virus HA)and the globular head domain of the cH7/3 chimeric influenzahemagglutinin polypeptide is the globular head domain of A/Canada/444/04(H7) HA (or the globular head domain of an A/Canada/444/04 (H7)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/3 chimeric influenza hemagglutinin polypeptide is the stem domainof A/harbor seal/Massachusetts/1/2011 (H3N8) HA (or the stem domain ofan A/harbor seal/Massachusetts/1/2011 (H3N8)-like influenza virus HA)and the globular head domain of the cH7/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/chicken/Jalisco/CPA1/2012 (H7) HA (or the globular head domain of anA/chicken/Jalisco/CPA1/2012 (H7)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH7/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA (or the stem domain of an A/harborseal/Massachusetts/1/2011 (H3N8)-like influenza virus HA) and theglobular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/mallard/Alberta/24/2001(H7) HA (or the globular head domain of an A/mallard/Alberta/24/2001(H7)-like influenza virus HA). In another specific embodiment, the stemdomain of a cH7/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/harbor seal/Massachusetts/1/2011 (H3N8) HA (or the stemdomain of an A/harbor seal/Massachusetts/1/2011 (H3N8)-like influenzavirus HA) and the globular head domain of the cH7/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Rhea/NC/39482/93 (H7) HA (or the globular head domain of anA/Rhea/NC/39482/93 (H7)-like influenza virus HA). In another specificembodiment, the stem domain of a cH7/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/harbor seal/Massachusetts/1/2011(H3N8) HA (or the stem domain of an A/harbor seal/Massachusetts/1/2011(H3N8)-like influenza virus HA) and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/mallard/Netherlands/12/2000 (H7) HA (or the globular headdomain of an A/mallard/Netherlands/12/2000 (H7)-like influenza virusHA).

In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA (or the stem domain of an A/Indiana/10/2011(H3N2)-like influenza virus HA). In another specific embodiment, thestem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide isthe stem domain of A/Indiana/10/2011 (H3N2) HA (or the stem domain of anA/Indiana/10/2011 (H3N2)-like influenza virus HA) and the globular headdomain of the cH7/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Netherlands/219/03 (H7) HA (or the globularhead domain of an A/Netherlands/219/03 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA (or the stem domain of an A/Indiana/10/2011(H3N2)-like influenza virus HA) and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Canada/504/04 (H7) HA (or the globular head domain of anA/Canada/504/04 (H7)-like influenza virus HA). In another specificembodiment, the stem domain of a cH7/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Indiana/10/2011 (H3N2) HA (or thestem domain of an A/Indiana/10/2011 (H3N2)-like influenza virus HA) andthe globular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Canada/444/04 (H7) HA (orthe globular head domain of an A/Canada/444/04 (H7)-like influenza virusHA). In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA (or the stem domain of an A/Indiana/10/2011(H3N2)-like influenza virus HA) and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/chicken/Jalisco/CPA1/2012 (H7) HA (or the globular headdomain of an A/chicken/Jalisco/CPA1/2012 (H7)-like influenza virus HA).In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA (or the stem domain of an A/Indiana/10/2011(H3N2)-like influenza virus HA) and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/mallard/Alberta/24/2001 (H7) HA (or the globular head domainof an A/mallard/Alberta/24/2001 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA (or the stem domain of an A/Indiana/10/2011(H3N2)-like influenza virus HA) and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Rhea/NC/39482/93 (H7) HA (or the globular head domain of anA/Rhea/NC/39482/93 (H7)-like influenza virus HA). In another specificembodiment, the stem domain of a cH7/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Indiana/10/2011 (H3N2) HA (or thestem domain of an A/Indiana/10/2011 (H3N2)-like influenza virus HA) andthe globular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/mallard/Netherlands/12/2000(H7) HA (or the globular head domain of an A/mallard/Netherlands/12/2000(H7)-like influenza virus HA).

In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA (or the stem domain of an A/Perth/16/2009(H3N2)-like influenza virus HA). In another specific embodiment, thestem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide isthe stem domain of A/Perth/16/2009 (H3N2) HA (or the stem domain of anA/Perth/16/2009 (H3N2)-like influenza virus HA) and the globular headdomain of the cH7/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Netherlands/219/03 (H7) HA (or the globularhead domain of an A/Netherlands/219/03 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA (or the stem domain of an A/Perth/16/2009(H3N2)-like influenza virus HA) and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Canada/504/04 (H7) HA (or the globular head domain of anA/Canada/504/04 (H7)-like influenza virus HA). In another specificembodiment, the stem domain of a cH7/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Perth/16/2009 (H3N2) HA (or the stemdomain of an A/Perth/16/2009 (H3N2)-like influenza virus HA) and theglobular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Canada/444/04 (H7) HA (orthe globular head domain of an A/Canada/444/04 (H7)-like influenza virusHA). In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA (or the stem domain of an A/Perth/16/2009(H3N2)-like influenza virus HA) and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/chicken/Jalisco/CPA1/2012 (H7) HA (or the globular headdomain of an A/chicken/Jalisco/CPA1/2012 (H7)-like influenza virus HA).In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA (or the stem domain of an A/Perth/16/2009(H3N2)-like influenza virus HA) and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/mallard/Alberta/24/2001 (H7) HA (or the globular head domainof an A/mallard/Alberta/24/2001 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA (or the stem domain of an A/Perth/16/2009(H3N2)-like influenza virus HA) and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Rhea/NC/39482/93 (H7) HA (or the globular head domain of anA/Rhea/NC/39482/93 (H7)-like influenza virus HA). In another specificembodiment, the stem domain of a cH7/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Perth/16/2009 (H3N2) HA (or the stemdomain of an A/Perth/16/2009 (H3N2)-like influenza virus HA) and theglobular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/mallard/Netherlands/12/2000(H7) HA (or the globular head domain of an A/mallard/Netherlands/12/2000(H7)-like influenza virus HA).

In specific embodiments, the stem domain of the hemagglutinin from aninfluenza virus of the H3 subtype of a cH7/3 chimeric influenzahemagglutinin polypeptide provided herein is from an H3 subtype that themajority of the population is naive to. In certain embodiments, the stemdomain of the hemagglutinin from an influenza virus of the H3 subtype ofa cH7/3 chimeric influenza hemagglutinin polypeptide provided herein isfrom an upcoming H3N2 vaccine strain, e.g., the H3N2 vaccine strain inuse in the year 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021,2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2032,2033, 2034, or 2035.

In a specific embodiment, a cH7/3 chimeric influenza hemagglutininpolypeptide does not comprise the globular head domain ofA/mallard/Alberta/24/2001 (H7). In another specific embodiment, a cH7/3chimeric influenza hemagglutinin polypeptide does not comprise the stemdomain of A/Perth/16/2009 (H3).

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza B virus and (ii) the globular headdomain of the hemagglutinin from an influenza virus of the H5 subtype(sometimes referred to herein as a “cH5/B chimeric influenzahemagglutinin polypeptide”).

In a specific embodiment, the stem domain of a cH5/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HA(or the stem domain of an B/Malaysia/2506/2004-like influenza virus HA).In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Malaysia/2506/2004 HA (or the stem domain of anB/Malaysia/2506/2004-like influenza virus HA) and the globular headdomain of the cH5/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Vietnam/1203/2004 (H5) HA (or the globularhead domain of an A/Vietnam/1203/2004 (H5)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Malaysia/2506/2004 HA (or the stem domain of anB/Malaysia/2506/2004-like influenza virus HA) and the globular headdomain of the cH5/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Indonesia/5/2005 (H5) HA (or the globular headdomain of an A/Indonesia/5/2005 (H5)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Malaysia/2506/2004 HA (or the stem domain of anB/Malaysia/2506/2004-like influenza virus HA) and the globular headdomain of the cH5/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Anhui/1/2005 (H5) HA (or the globular headdomain of an A/Anhui/1/2005 (H5)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH5/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HA(or the stem domain of an B/Malaysia/2506/2004-like influenza virus HA)and the globular head domain of the cH5/B chimeric influenzahemagglutinin polypeptide is the globular head domain of A/Bar headedgoose/Quinghai/1A/2005 (H5) HA (or the globular head domain of an A/Barheaded goose/Quinghai/1A/2005 (H5)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH5/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HA(or the stem domain of an B/Malaysia/2506/2004-like influenza virus HA)and the globular head domain of the cH5/B chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/turkey/Turkey/1/2005 (H5) HA (or the globular head domain of anA/turkey/Turkey/1/2005 (H5)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH5/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HA(or the stem domain of an B/Malaysia/2506/2004-like influenza virus HA)and the globular head domain of the cH5/B chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Whooperswan/Mongolia/244/2005 (H5) HA (or the globular head domain ofan A/Whooperswan/Mongolia/244/2005 (H5)-like influenza virus HA).

In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH5/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA) and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/Vietnam/1203/2004 (H5) HA (or the globular head domain of anA/Vietnam/1203/2004 (H5)-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Florida/4/2006 HA (or the stemdomain of an B/Florida/4/2006-like influenza virus HA) and the globularhead domain of the cH5/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Indonesia/5/2005 (H5) HA (or the globularhead domain of an A/Indonesia/5/2005 (H5)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA) and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/Anhui/1/2005 (H5) HA (or the globular head domain of an A/Anhui/1/2005(H5)-like influenza virus HA). In another specific embodiment, the stemdomain of a cH5/B chimeric influenza hemagglutinin polypeptide is thestem domain of B/Florida/4/2006 HA (or the stem domain of anB/Florida/4/2006-like influenza virus HA) and the globular head domainof the cH5/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA (orthe globular head domain of an A/Bar headed goose/Quinghai/1A/2005(H5)-like influenza virus HA). In another specific embodiment, the stemdomain of a cH5/B chimeric influenza hemagglutinin polypeptide is thestem domain of B/Florida/4/2006 HA (or the stem domain of anB/Florida/4/2006-like influenza virus HA) and the globular head domainof the cH5/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/turkey/Turkey/1/2005 (H5) HA (or the globularhead domain of an A/turkey/Turkey/1/2005 (H5)-like influenza virus HA).In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA) and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/Whooperswan/Mongolia/244/2005 (H5) HA (or the globular head domain ofan A/Whooperswan/Mongolia/244/2005 (H5)-like influenza virus HA).

In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA (or the stem domain of an B/Wisconsin/1/2010-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH5/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Wisconsin/1/2010 HA (or the stem domain of anB/Wisconsin/1/2010-like influenza virus HA) and the globular head domainof the cH5/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Vietnam/1203/2004 (H5) HA (or the globularhead domain of an A/Vietnam/1203/2004 (H5)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA (or the stem domain of an B/Wisconsin/1/2010-likeinfluenza virus HA) and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/Indonesia/5/2005 (H5) HA (or the globular head domain of anA/Indonesia/5/2005 (H5)-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Wisconsin/1/2010 HA (or the stemdomain of an B/Wisconsin/1/2010-like influenza virus HA) and theglobular head domain of the cH5/B chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Anhui/1/2005 (H5) HA (orthe globular head domain of an A/Anhui/1/2005 (H5)-like influenza virusHA). In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA (or the stem domain of an B/Wisconsin/1/2010-likeinfluenza virus HA) and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain of A/Barheaded goose/Quinghai/1A/2005 (H5) HA (or the globular head domain of anA/Bar headed goose/Quinghai/1A/2005 (H5)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA (or the stem domain of an B/Wisconsin/1/2010-likeinfluenza virus HA) and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/turkey/Turkey/1/2005 (H5) HA (or the globular head domain of anA/turkey/Turkey/1/2005 (H5)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH5/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Wisconsin/1/2010 HA(or the stem domain of an B/Wisconsin/1/2010-like influenza virus HA)and the globular head domain of the cH5/B chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Whooperswan/Mongolia/244/2005 (H5) HA (or the globular head domain ofan A/Whooperswan/Mongolia/244/2005 (H5)-like influenza virus HA).

In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA (or the stem domain of an B/Brisbane/60/2008-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH5/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Brisbane/60/2008 HA (or the stem domain of anB/Brisbane/60/2008-like influenza virus HA) and the globular head domainof the cH5/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Vietnam/1203/2004 (H5) HA (or the globularhead domain of an A/Vietnam/1203/2004 (H5)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA (or the stem domain of an B/Brisbane/60/2008-likeinfluenza virus HA) and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/Indonesia/5/2005 (H5) HA (or the globular head domain of anA/Indonesia/5/2005 (H5)-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Brisbane/60/2008 HA (or the stemdomain of an B/Brisbane/60/2008-like influenza virus HA) and theglobular head domain of the cH5/B chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Anhui/1/2005 (H5) HA (orthe globular head domain of an A/Anhui/1/2005 (H5)-like influenza virusHA). In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA (or the stem domain of an B/Brisbane/60/2008-likeinfluenza virus HA) and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain of A/Barheaded goose/Quinghai/1A/2005 (H5) HA (or the globular head domain of anA/Bar headed goose/Quinghai/1A/2005 (H5)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA (or the stem domain of an B/Brisbane/60/2008-likeinfluenza virus HA) and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/turkey/Turkey/1/2005 (H5) HA (or the globular head domain of anA/turkey/Turkey/1/2005 (H5)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH5/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Brisbane/60/2008 HA(or the stem domain of an B/Brisbane/60/2008-like influenza virus HA)and the globular head domain of the cH5/B chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Whooperswan/Mongolia/244/2005 (H5) HA (or the globular head domain ofan A/Whooperswan/Mongolia/244/2005 (H5)-like influenza virus HA).

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza B virus and (ii) the globular headdomain of the hemagglutinin from an influenza virus of the H7 subtype(sometimes referred to herein as a “cH7/B chimeric influenzahemagglutinin polypeptide”).

In a specific embodiment, the stem domain of a cH7/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HA(or the stem domain of an B/Malaysia/2506/2004-like influenza virus HA).In another specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Malaysia/2506/2004 HA (or the stem domain of anB/Malaysia/2506/2004-like influenza virus HA) and the globular headdomain of the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Netherlands/219/03 (H7) HA (or the globularhead domain of an A/Netherlands/219/03 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Malaysia/2506/2004 HA (or the stem domain of anB/Malaysia/2506/2004-like influenza virus HA) and the globular headdomain of the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Canada/504/04 (H7) HA (or the globular headdomain of an A/Canada/504/04 (H7)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH7/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HA(or the stem domain of an B/Malaysia/2506/2004-like influenza virus HA)and the globular head domain of the cH7/B chimeric influenzahemagglutinin polypeptide is the globular head domain of A/Canada/444/04(H7) HA (or the globular head domain of an A/Canada/444/04 (H7)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Malaysia/2506/2004 HA (or the stem domain of anB/Malaysia/2506/2004-like influenza virus HA) and the globular headdomain of the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/chicken/Jalisco/CPA1/2012 (H7) HA (or theglobular head domain of an A/chicken/Jalisco/CPA1/2012 (H7)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Malaysia/2506/2004 HA (or the stem domain of anB/Malaysia/2506/2004-like influenza virus HA) and the globular headdomain of the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/mallard/Alberta/24/2001 (H7) HA (or theglobular head domain of an A/mallard/Alberta/24/2001 (H7)-like influenzavirus HA). In another specific embodiment, the stem domain of a cH7/Bchimeric influenza hemagglutinin polypeptide is the stem domain ofB/Malaysia/2506/2004 HA (or the stem domain of anB/Malaysia/2506/2004-like influenza virus HA) and the globular headdomain of the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Rhea/NC/39482/93 (H7) HA (or the globular headdomain of an A/Rhea/NC/39482/93 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Malaysia/2506/2004 HA (or the stem domain of anB/Malaysia/2506/2004-like influenza virus HA) and the globular headdomain of the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/mallard/Massachusetts/12/2000 (H7) HA (or theglobular head domain of an A/mallard/Massachusetts/12/2000 (H7)-likeinfluenza virus HA).

In another specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA) and the globular head domain of the cH7/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/Netherlands/219/03 (H7) HA (or the globular head domain of anA/Netherlands/219/03 (H7)-like influenza virus HA). In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Florida/4/2006 HA (or the stemdomain of an B/Florida/4/2006-like influenza virus HA) and the globularhead domain of the cH7/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Canada/504/04 (H7) HA (or the globularhead domain of an A/Canada/504/04 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA) and the globular head domain of the cH7/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/Canada/444/04 (H7) HA (or the globular head domain of anA/Canada/444/04 (H7)-like influenza virus HA). In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Florida/4/2006 HA (or the stemdomain of an B/Florida/4/2006-like influenza virus HA) and the globularhead domain of the cH7/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/chicken/Jalisco/CPA1/2012 (H7) HA (or theglobular head domain of an A/chicken/Jalisco/CPA1/2012 (H7)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA) and the globular head domain of the cH7/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/mallard/Alberta/24/2001 (H7) HA (or the globular head domain of anA/mallard/Alberta/24/2001 (H7)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH7/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Florida/4/2006 HA (orthe stem domain of an B/Florida/4/2006-like influenza virus HA) and theglobular head domain of the cH7/B chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Rhea/NC/39482/93 (H7) HA(or the globular head domain of an A/Rhea/NC/39482/93 (H7)-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA) and the globular head domain of the cH7/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/mallard/Massachusetts/12/2000 (H7) HA (or the globular head domain ofan A/mallard/Massachusetts/12/2000 (H7)-like influenza virus HA).

In another specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA (or the stem domain of an B/Wisconsin/1/2010-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Wisconsin/1/2010 HA (or the stem domain of anB/Wisconsin/1/2010-like influenza virus HA) and the globular head domainof the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Netherlands/219/03 (H7) HA (or the globularhead domain of an A/Netherlands/219/03 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA (or the stem domain of an B/Wisconsin/1/2010-likeinfluenza virus HA) and the globular head domain of the cH7/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/Canada/504/04 (H7) HA (or the globular head domain of anA/Canada/504/04 (H7)-like influenza virus HA). In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Wisconsin/1/2010 HA (or the stemdomain of an B/Wisconsin/1/2010-like influenza virus HA) and theglobular head domain of the cH7/B chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Canada/444/04 (H7) HA (orthe globular head domain of an A/Canada/444/04 (H7)-like influenza virusHA). In another specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA (or the stem domain of an B/Wisconsin/1/2010-likeinfluenza virus HA) and the globular head domain of the cH7/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/chicken/Jalisco/CPA1/2012 (H7) HA (or the globular head domain of anA/chicken/Jalisco/CPA1/2012 (H7)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH7/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Wisconsin/1/2010 HA(or the stem domain of an B/Wisconsin/1/2010-like influenza virus HA)and the globular head domain of the cH7/B chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/mallard/Alberta/24/2001 (H7) HA (or the globular head domain of anA/mallard/Alberta/24/2001 (H7)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH7/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Wisconsin/1/2010 HA(or the stem domain of an B/Wisconsin/1/2010-like influenza virus HA)and the globular head domain of the cH7/B chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Rhea/NC/39482/93 (H7) HA (or the globular head domain of anA/Rhea/NC/39482/93 (H7)-like influenza virus HA). In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Wisconsin/1/2010 HA (or the stemdomain of an B/Wisconsin/1/2010-like influenza virus HA) and theglobular head domain of the cH7/B chimeric influenza hemagglutininpolypeptide is the globular head domain ofA/mallard/Massachusetts/12/2000 (H7) HA (or the globular head domain ofan A/mallard/Massachusetts/12/2000 (H7)-like influenza virus HA).

In another specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA (or the stem domain of an B/Brisbane/60/2008-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cH7/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Brisbane/60/2008 HA (or the stem domain of anB/Brisbane/60/2008-like influenza virus HA) and the globular head domainof the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Netherlands/219/03 (H7) HA (or the globularhead domain of an A/Netherlands/219/03 (H7)-like influenza virus HA). Inanother specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA (or the stem domain of an B/Brisbane/60/2008-likeinfluenza virus HA) and the globular head domain of the cH7/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/Canada/504/04 (H7) HA (or the globular head domain of anA/Canada/504/04 (H7)-like influenza virus HA). In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Brisbane/60/2008 HA (or the stemdomain of an B/Brisbane/60/2008-like influenza virus HA) and theglobular head domain of the cH7/B chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Canada/444/04 (H7) HA (orthe globular head domain of an A/Canada/444/04 (H7)-like influenza virusHA). In another specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA (or the stem domain of an B/Brisbane/60/2008-likeinfluenza virus HA) and the globular head domain of the cH7/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/chicken/Jalisco/CPA1/2012 (H7) HA (or the globular head domain of anA/chicken/Jalisco/CPA1/2012 (H7)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH7/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Brisbane/60/2008 HA(or the stem domain of an B/Brisbane/60/2008-like influenza virus HA)and the globular head domain of the cH7/B chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/mallard/Alberta/24/2001 (H7) HA (or the globular head domain of anA/mallard/Alberta/24/2001 (H7)-like influenza virus HA). In anotherspecific embodiment, the stem domain of a cH7/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Brisbane/60/2008 HA(or the stem domain of an B/Brisbane/60/2008-like influenza virus HA)and the globular head domain of the cH7/B chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Rhea/NC/39482/93 (H7) HA (or the globular head domain of anA/Rhea/NC/39482/93 (H7)-like influenza virus HA). In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Brisbane/60/2008 HA (or the stemdomain of an B/Brisbane/60/2008-like influenza virus HA) and theglobular head domain of the cH7/B chimeric influenza hemagglutininpolypeptide is the globular head domain ofA/mallard/Massachusetts/12/2000 (H7) HA (or the globular head domain ofan A/mallard/Massachusetts/12/2000 (H7)-like influenza virus HA).

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza B virus and (ii) the globular headdomain of the hemagglutinin from a different influenza B virus strain(sometimes referred to herein as a “cB/B chimeric influenzahemagglutinin polypeptide”).

In a specific embodiment, the stem domain of a cB/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HA(or the stem domain of an B/Malaysia/2506/2004-like influenza virus HA).In another specific embodiment, the stem domain of a cB/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Malaysia/2506/2004 HA (or the stem domain of anB/Malaysia/2506/2004-like influenza virus HA) and the globular headdomain of the cB/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of B/Lee/1940 HA (or the globular head domain of anB/Lee/1940-like influenza virus HA). In another specific embodiment, thestem domain of a cB/B chimeric influenza hemagglutinin polypeptide isthe stem domain of B/Malaysia/2506/2004 HA (or the stem domain of anB/Malaysia/2506/2004-like influenza virus HA) and the globular headdomain of the cB/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of B/seal/Netherlands/1/99 HA (or the globular headdomain of a B/seal/Netherlands/1/99-like influenza virus).

In another specific embodiment, the stem domain of a cB/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cB/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA) and the globular head domain of the cB/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofB/Lee/1940 HA (or the globular head domain of a B/Lee/1940-likeinfluenza virus). In another specific embodiment, the stem domain of acB/B chimeric influenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA (or the stem domain of an B/Florida/4/2006-likeinfluenza virus HA) and the globular head domain of the cB/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofB/seal/Netherlands/1/99 HA (or the globular head domain of aB/seal/Netherlands/1/99-like influenza virus).

In another specific embodiment, the stem domain of a cB/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA (or the stem domain of an B/Wisconsin/1/2010-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cB/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Wisconsin/1/2010 HA (or the stem domain of anB/Wisconsin/1/2010-like influenza virus HA) and the globular head domainof the cB/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of B/Lee/1940 HA (or the globular head domain of aB/Lee/1940-like influenza virus). In another specific embodiment, thestem domain of a cB/B chimeric influenza hemagglutinin polypeptide isthe stem domain of B/Wisconsin/1/2010 HA (or the stem domain of anB/Wisconsin/1/2010-like influenza virus HA) and the globular head domainof the cB/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of B/seal/Netherlands/1/99 HA (or the globular head domainof a B/seal/Netherlands/1/99-like influenza virus).

In another specific embodiment, the stem domain of a cB/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA (or the stem domain of an B/Brisbane/60/2008-likeinfluenza virus HA). In another specific embodiment, the stem domain ofa cB/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Brisbane/60/2008 HA (or the stem domain of anB/Brisbane/60/2008-like influenza virus HA) and the globular head domainof the cB/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of B/Lee/1940 HA (or the globular head domain of aB/Lee/1940-like influenza virus). In another specific embodiment, thestem domain of a cB/B chimeric influenza hemagglutinin polypeptide isthe stem domain of B/Brisbane/60/2008 HA (or the stem domain of anB/Brisbane/60/2008-like influenza virus HA) and the globular head domainof the cB/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of B/seal/Netherlands/1/99 HA (or the globular head domainof a B/seal/Netherlands/1/99-like influenza virus).

In specific embodiments, the chimeric influenza hemagglutininpolypeptides described herein are soluble, e.g., are soluble incompositions, e.g., the compositions described herein. Exemplary methodsfor generating soluble chimeric influenza hemagglutinin polypeptides aredescribed in Section 6.6.1.2, infra.

When designing the foregoing chimeric influenza HA polypeptides, careshould be taken to maintain the stability of the resulting protein. Inthis regard, in certain embodiments, it is recommended that for chimericinfluenza hemagglutinin polypeptides comprising stem domains frominfluenza A viruses, the cysteine residues identified as Ap and Aq inFIG. 1 be maintained since they contribute to the stability of the HAstalk as discussed in more detail in Section 5.1 infra. For example, forthe best stability, it is preferred to “swap” the HA globular domain asa whole (between the Ap and Aq cysteine residues as shown in FIG. 1)since the resulting conformation would be closest to the nativestructure. Similarly, chimeric influenza hemagglutinin polypeptidescomprising the stem domains of influenza B viruses may utilize cysteinespresent in the globular head domains of the influenza A virus from whichthe globular head domain of the chimeric influenza hemagglutininpolypeptide is obtained (see, e.g., FIG. 36).

In another aspect, provided herein are immunogenic compositions (e.g.,vaccine formulations) comprising one, two, or more of the chimericinfluenza hemagglutinin polypeptides described herein. In certainembodiments, the immunogenic compositions (e.g., vaccine formulations)provided herein may comprise chimeric influenza hemagglutininpolypeptide(s) described herein, influenza viruses (e.g., live or killedvirus) that comprise a chimeric influenza hemagglutinin polypeptide(s)described herein or a genome engineered to encode chimeric influenzahemagglutinin polypeptide(s) described herein; or vectors or cells thatcomprise a chimeric influenza hemagglutinin polypeptide(s) describedherein or a genome engineered to encode chimeric influenza hemagglutininpolypeptide(s) described herein. In certain embodiments, the immunogeniccompositions provided herein may comprise (i) a cH5/1 chimeric influenzahemagglutinin polypeptide described herein, a cH5/3 chimeric influenzahemagglutinin polypeptide described herein, a cH7/3 chimeric influenzahemagglutinin polypeptide described herein, a cH5/B chimeric influenzahemagglutinin polypeptide described herein, a cH7/B chimeric influenzahemagglutinin polypeptide described herein, or a cHB/B chimericinfluenza hemagglutinin polypeptide described herein; (ii) a combinationof a cH5/1 chimeric influenza hemagglutinin polypeptide described hereinand a cH5/3 chimeric influenza hemagglutinin polypeptide describedherein; or a combination of a cH5/1 chimeric influenza hemagglutininpolypeptide described herein and a cH7/3 chimeric influenzahemagglutinin polypeptide described herein; (iii) a combination of acH5/1 chimeric influenza hemagglutinin polypeptide described herein anda cH5/3 chimeric influenza hemagglutinin polypeptide described hereinand either of a cH5/B, a cH7/B, or a cB/B chimeric influenzahemagglutinin polypeptide described herein; or (iv) a combination of acH5/1 chimeric influenza hemagglutinin polypeptide described herein anda cH7/3 chimeric influenza hemagglutinin polypeptide described hereinand either of a cH5/B, a cH7/B, or a cB/B chimeric influenzahemagglutinin polypeptide described herein.

In a specific embodiment, provided herein are vaccine formulationscomprising one or more of the chimeric influenza hemagglutininpolypeptides described herein. In a specific embodiment, provided hereinis a monovalent vaccine comprising one of the chimeric influenzahemagglutinin polypeptides described herein. In another specificembodiment, provided herein is a bivalent vaccine comprising two of thechimeric influenza hemagglutinin polypeptides described herein (i.e.,two distinct chimeric influenza hemagglutinin polypeptides). In anotherspecific embodiment, provided herein is a trivalent vaccine comprisingthree of the chimeric influenza hemagglutinin polypeptides describedherein (i.e., three distinct chimeric influenza hemagglutininpolypeptides).

The vaccine formulations provided herein may comprise the chimericinfluenza hemagglutinin polypeptides described herein in any form. Forexample, the vaccine formulations provided herein may comprise subunitvaccines comprising one or more of the chimeric influenza hemagglutininpolypeptides described herein (e.g., compositions comprising chimericinfluenza hemagglutinin polypeptides, e.g., soluble chimeric influenzahemagglutinin polypeptides); live influenza viruses (e.g., liveattenuated influenza viruses) that express one or more of the chimericinfluenza hemagglutinin polypeptides described herein; live influenzaviruses (e.g., live attenuated influenza viruses) comprising a genomethat encodes one or more of the chimeric influenza hemagglutininpolypeptides described herein; killed influenza viruses that compriseone or more of the chimeric influenza hemagglutinin polypeptidesdescribed herein; killed influenza viruses comprising a genome thatencodes one or more of the chimeric influenza hemagglutinin polypeptidesdescribed herein; virus/viral-like particles (“VLPs”) that contain oneor more of the chimeric influenza hemagglutinin polypeptides describedherein; split virus vaccines, wherein said virus expresses one or moreof the chimeric influenza hemagglutinin polypeptides described hereinand/or comprises a genome that encodes one or more of the chimericinfluenza hemagglutinin polypeptides described herein; viral expressionvectors (e.g., non-influenza virus expression vectors) that express oneor more of the chimeric influenza hemagglutinin polypeptides describedherein; and bacterial expression vectors that express one or more of thechimeric influenza hemagglutinin polypeptides described herein.

The vaccine formulations described herein can elicit highly potent andbroadly neutralizing antibodies against the HA stem domain of thechimeric influenza hemagglutinin polypeptides. Such “universal” vaccinescan be used to induce and/or boost cross-protective immune responsesacross influenza virus subtypes.

In another aspect, provided herein are methods of immunizing a subjectagainst an influenza virus disease or infection comprising administeringto the subject a composition comprising one or more of the chimericinfluenza hemagglutinin (HA) polypeptides described herein or a vaccineformulation described herein. In certain embodiments, a subject isprimed with a first composition comprising one or more of the chimericinfluenza hemagglutinin (HA) polypeptides described herein or a vaccineformulation described herein, and later boosted with the same or adifferent composition (e.g., a composition comprising a differentchimeric influenza hemagglutinin (HA) polypeptide; a compositioncomprising the same chimeric influenza hemagglutinin (HA) polypeptidebut in a different context (e.g., the first composition comprises asubunit vaccine comprising a chimeric influenza hemagglutinin (HA)polypeptide and the different composition comprises a viral vector thatcomprises the same chimeric influenza hemagglutinin (HA) polypeptide),or a composition comprising a different chimeric influenza hemagglutinin(HA) polypeptide in a different context) comprising one or more of thechimeric influenza hemagglutinin (HA) polypeptides described herein or avaccine formulation described herein. The subject may be boosted once,or more than once with a composition comprising one or more of thechimeric influenza hemagglutinin (HA) polypeptides described herein or avaccine formulation described herein. In certain embodiments, when thesubject is boosted more than once, the first and the second boosts arewith different compositions comprising one or more of the chimericinfluenza hemagglutinin (HA) polypeptides described herein or a vaccineformulation described herein, and each boost comprises a differentcomposition than the composition comprising one or more of the chimericinfluenza hemagglutinin (HA) polypeptides described herein or a vaccineformulation described herein used to prime the subject.

In a specific embodiment, provided herein is a method of immunizing asubject (e.g., a human subject) against influenza virus comprisingadministering to the subject a first dose of an effective amount of achimeric influenza hemagglutinin (HA) polypeptide described herein, avector described herein, an immunogenic composition described herein, ora vaccine formulation described herein and administering to the subjecta second dose of an effective amount of a chimeric influenzahemagglutinin (HA) polypeptide described herein, a vector describedherein, an immunogenic composition described herein, or a vaccineformulation described herein 30 days to 6 months after the subject hasreceived the first dose, wherein (i) the chimeric influenzahemagglutinin (HA) polypeptide or the chimeric influenza hemagglutinin(HA) polypeptide of the vector, the immunogenic composition, or vaccineformulation in the first and second doses are the same or different(e.g., the globular head of the chimeric influenza hemagglutinin (HA)polypeptide administered in the first dose is different than theglobular head of the chimeric influenza hemagglutinin (HA) polypeptideadministered in the second dose); and/or (ii) the type of immunogeniccomposition or vector or vaccine formulation administered in both dosesare the same or different. In certain embodiments, the method comprisesadministering to the subject a third dose of an effective amount of achimeric influenza hemagglutinin (HA) polypeptide described herein, avector described herein, an immunogenic composition described herein, ora vaccine formulation described herein 30 days to 6 months after thesubject has received the second dose, wherein (i) the chimeric influenzahemagglutinin (HA) polypeptide or the chimeric influenza hemagglutinin(HA) polypeptide of the vector, the immunogenic composition, or vaccineformulation is the same or different than the chimeric influenzahemagglutinin (HA) polypeptide in the first and/or second dose; and (ii)the type of immunogenic composition or vector or vaccine formulationadministered in both doses are the same or different. In certainembodiments, two, three, or more chimeric influenza hemagglutinin (HA)polypeptides are administered as part of the first, second, and/or thirddoses, wherein each chimeric HA polypeptide in a dose is different fromeach other. In some embodiments, the first, second, and/or third dose ofthe vector, the immunogenic composition, or vaccine formulationcomprises two, three, or more chimeric influenza hemagglutinin (HA)polypeptides, wherein each chimeric influenza hemagglutinin (HA)polypeptide in the vector, the immunogenic composition, or vaccineformulation administered in a dose is different from each other (e.g.,the globular head of the chimeric influenza hemagglutinin (HA)polypeptide administered in the first dose is different than theglobular head of the chimeric influenza hemagglutinin (HA) polypeptideadministered in the second dose, etc.).

In another specific embodiment, provided herein is a method ofimmunizing a 1-5 year old human subject against influenza viruscomprising administering to the subject a first dose of an effectiveamount a chimeric influenza hemagglutinin (HA) polypeptide describedherein, a vector described herein, an immunogenic composition describedherein, or a vaccine formulation described herein and administering tothe subject a second dose of an effective amount of a chimeric influenzahemagglutinin (HA) polypeptide described herein, a vector describedherein, an immunogenic composition described herein, or a vaccineformulation described herein 30 days to 6 months after the subject hasreceived the first dose, wherein (i) the chimeric influenzahemagglutinin (HA) polypeptide or the chimeric influenza hemagglutinin(HA) polypeptide of the vector, the immunogenic composition, or vaccineformulation in the first and second doses are the same or different(e.g., the globular head of the chimeric influenza hemagglutinin (HA)polypeptide administered in the first dose is different than theglobular head of the chimeric influenza hemagglutinin (HA) polypeptideadministered in the second dose); and/or (ii) the type of immunogeniccomposition or vector or vaccine formulation administered in both dosesare the same or different. In certain embodiments, the method comprisesadministering to the subject a third dose of an effective amount of achimeric influenza hemagglutinin (HA) polypeptide described herein, avector described herein, an immunogenic composition described herein, ora vaccine formulation described herein 30 days to 6 months after thesubject has received the second dose, wherein (i) the chimeric influenzahemagglutinin (HA) polypeptide or the chimeric influenza hemagglutinin(HA) polypeptide of the vector, the immunogenic composition, or vaccineformulation in the first and second doses are the same or different(e.g., the globular head of the chimeric influenza hemagglutinin (HA)polypeptide administered in the first dose is different than theglobular head of the chimeric influenza hemagglutinin (HA) polypeptideadministered in the second dose); and/or (ii) the type of immunogeniccomposition or vector or vaccine formulation administered in both dosesare the same or different. In certain embodiments, two, three, or morechimeric influenza hemagglutinin (HA) polypeptides are administered aspart of the first, second, and/or third doses, wherein each chimeric HApolypeptide in a dose is different from each other. In some embodiments,the first, second, and/or third dose of the vector, the immunogeniccomposition, or vaccine formulation comprises two, three, or morechimeric influenza hemagglutinin (HA) polypeptides, wherein eachchimeric influenza hemagglutinin (HA) polypeptide in the vector, theimmunogenic composition, or vaccine formulation administered in a doseis different from each other (e.g., the globular head of the chimericinfluenza hemagglutinin (HA) polypeptide administered in the first doseis different than the globular head of the chimeric influenzahemagglutinin (HA) polypeptide administered in the second dose, etc.).

In another aspect, provided herein are kits comprising one or more ofthe chimeric influenza hemagglutinin (HA) polypeptides described hereinor a vaccine formulation described herein. The kits provided herein mayfurther comprise one or more additional components, e.g., an antibodythat specifically binds one or more of the chimeric influenzahemagglutinin (HA) polypeptides provided in the kit.

The working Examples (e.g., Section 6, Examples) demonstrate, interalia, the production of constructs encoding chimeric influenza HApolypeptides comprising an HA stem domain and displaying a heterologousHA globular head domain, and the production of stable chimeric HApolypeptides from these constructs which are cross-reactive withantibodies to both the stem domain and the head domain. The workingExamples also illustrate the use of such constructs in the generation ofa protective immune response in subjects against multiple differentstrains and subtypes of influenza virus, i.e., the Examples demonstratethat the chimeric influenza HA polypeptides described herein can be usedas a universal influenza vaccine.

3.1 Terminology

The terms “about” or “approximate,” when used in reference to an aminoacid position refer to the particular amino acid position in a sequenceor any amino acid that is within five, four, three, two, or one residuesof that amino acid position, either in an N-terminal direction or aC-terminal direction.

As used herein, the term “about” or “approximately” when used inconjunction with a number refers to any number within 1, 5 or 10% of thereferenced number. In certain embodiments, the term “about” encompassesthe exact number recited.

As used herein, the term “fragment” in the context of a nucleic acidsequence refers to a nucleotide sequence comprising a portion ofconsecutive nucleotides from a parent sequence. In a specificembodiment, the term refers to a nucleotide sequence of 5 to 15, 5 to25, 10 to 30, to 30, 10 to 60, 25 to 100, 150 to 300 or more consecutivenucleotides from a parent sequence. In another embodiment, the termrefers to a nucleotide sequence of at least 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 125, 150, 175,200, 250, 275, 300, 325, 350, 375, 400, 425, 450 or 475 consecutivenucleotides of a parent sequence.

As used herein, the term “fragment” in the context of an amino acidsequence refers to an amino acid sequence comprising a portion ofconsecutive amino acid residues from a parent sequence. In a specificembodiment, the term refers to an amino acid sequence of 2 to 30, 5 to30, 10 to 60, 25 to 100, 150 to 300 or more consecutive amino acidresidues from a parent sequence. In another embodiment, the term refersto an amino acid sequence of at least 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 125, 150, 175, or 200consecutive amino acid residues of a parent sequence.

As used herein, the terms “disease” and “disorder” are usedinterchangeably to refer to a condition in a subject. In specificembodiments, a term “disease” refers to the pathological state resultingfrom the presence of the virus in a cell or a subject, or by theinvasion of a cell or subject by the virus. In certain embodiments, thecondition is a disease in a subject, the severity of which is decreasedby inducing an immune response in the subject through the administrationof an immunogenic composition.

As used herein, the term “effective amount” in the context ofadministering a therapy to a subject refers to the amount of a therapywhich has a prophylactic and/or therapeutic effect(s). In certainembodiments, an “effective amount” in the context of administration of atherapy to a subject refers to the amount of a therapy which issufficient to achieve one, two, three, four, or more of the followingeffects: (i) reduce or ameliorate the severity of an influenza virusinfection, disease or symptom associated therewith; (ii) reduce theduration of an influenza virus infection, disease or symptom associatedtherewith; (iii) prevent the progression of an influenza virusinfection, disease or symptom associated therewith; (iv) causeregression of an influenza virus infection, disease or symptomassociated therewith; (v) prevent the development or onset of aninfluenza virus infection, disease or symptom associated therewith; (vi)prevent the recurrence of an influenza virus infection, disease orsymptom associated therewith; (vii) reduce or prevent the spread of aninfluenza virus from one cell to another cell, one tissue to anothertissue, or one organ to another organ; (ix) prevent or reduce the spreadof an influenza virus from one subject to another subject; (x) reduceorgan failure associated with an influenza virus infection; (xi) reducehospitalization of a subject; (xii) reduce hospitalization length;(xiii) increase the survival of a subject with an influenza virusinfection or disease associated therewith; (xiv) eliminate an influenzavirus infection or disease associated therewith; (xv) inhibit or reduceinfluenza virus replication; (xvi) inhibit or reduce the entry of aninfluenza virus into a host cell(s); (xviii) inhibit or reducereplication of the influenza virus genome; (xix) inhibit or reducesynthesis of influenza virus proteins; (xx) inhibit or reduce assemblyof influenza virus particles; (xxi) inhibit or reduce release ofinfluenza virus particles from a host cell(s); (xxii) reduce influenzavirus titer; and/or (xxiii) enhance or improve the prophylactic ortherapeutic effect(s) of another therapy.

In certain embodiments, the effective amount does not result in completeprotection from an influenza virus disease, but results in a lower titeror reduced number of influenza viruses compared to an untreated subject.In certain embodiments, the effective amount results in a 0.5 fold, 1fold, 2 fold, 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25fold, 50 fold, 75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200fold, 300 fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greaterreduction in titer of influenza virus relative to an untreated subject.In some embodiments, the effective amount results in a reduction intiter of influenza virus relative to an untreated subject ofapproximately 1 log or more, approximately 2 logs or more, approximately3 logs or more, approximately 4 logs or more, approximately 5 logs ormore, approximately 6 logs or more, approximately 7 logs or more,approximately 8 logs or more, approximately 9 logs or more,approximately 10 logs or more, 1 to 3 logs, 1 to 5 logs, 1 to 8 logs, 1to 9 logs, 2 to 10 logs, 2 to 5 logs, 2 to 7 logs, 2 logs to 8 logs, 2to 9 logs, 2 to 10 logs 3 to 5 logs, 3 to 7 logs, 3 to 8 logs, 3 to 9logs, 4 to 6 logs, 4 to 8 logs, 4 to 9 logs, 5 to 6 logs, 5 to 7 logs, 5to 8 logs, 5 to 9 logs, 6 to 7 logs, 6 to 8 logs, 6 to 9 logs, 7 to 8logs, 7 to 9 logs, or 8 to 9 logs. Benefits of a reduction in the titer,number or total burden of influenza virus include, but are not limitedto, less severe symptoms of the infection, fewer symptoms of theinfection and a reduction in the length of the disease associated withthe infection.

“Hemagglutinin” and “HA” refer to any hemagglutinin known to those ofskill in the art. In certain embodiments, the hemagglutinin is influenzahemagglutinin, such as an influenza A hemagglutinin, an influenza Bhemagglutinin, or an influenza C hemagglutinin. A typical hemagglutinincomprises domains known to those of skill in the art including a signalpeptide (optional herein), a stem domain, a globular head domain, aluminal domain (optional herein), a transmembrane domain (optionalherein) and a cytoplasmic domain (optional herein). In certainembodiments, a hemagglutinin consists of a single polypeptide chain,such as HA0. In certain embodiments, a hemagglutinin consists of morethan one polypeptide chain in quaternary association, e.g. HA1 and HA2.Those of skill in the art will recognize that an immature HA0 might becleaved to release a signal peptide (approximately 20 amino acids)yielding a mature hemagglutinin HA0. A hemagglutinin HA0 might becleaved at another site to yield HA1 polypeptide (approximately 320amino acids, including the globular head domain and a portion of thestem domain) and HA2 polypeptide (approximately 220 amino acids,including the remainder of the stem domain, a luminal domain, atransmembrane domain and a cytoplasmic domain). In certain embodiments,a hemagglutinin comprises a signal peptide, a transmembrane domain and acytoplasmic domain. In certain embodiments, a hemagglutinin lacks asignal peptide, i.e. the hemagglutinin is a mature hemagglutinin. Incertain embodiments, a hemagglutinin lacks a transmembrane domain orcytoplasmic domain, or both. As used herein, the terms “hemagglutinin”and “HA” encompass hemagglutinin polypeptides that are modified bypost-translational processing such as signal peptide cleavage, disulfidebond formation, glycosylation (e.g., N-linked glycosylation), proteasecleavage and lipid modification (e.g. S-palmitoylation).

As used herein, the terms “chimeric influenza virus hemagglutininpolypeptide,” “chimeric influenza virus HA polypeptide,” “chimerichemagglutinin polypeptide” and “chimeric influenza hemagglutininpolypeptide” refer to an influenza hemagglutinin that comprises aninfluenza virus hemagglutinin stem domain and an influenza virushemagglutinin globular head domain, wherein the influenza virushemagglutinin head domain is heterologous to the influenza virushemagglutinin stem domain (i.e., the globular head domain of thechimeric influenza virus hemagglutinin polypeptide is from a differentstrain or subtype of influenza virus than the stem domain of thechimeric influenza virus hemagglutinin polypeptide).

“HA1 N-terminal stem segment” refers to a polypeptide segment thatcorresponds to the amino-terminal portion of the stem domain of aninfluenza hemagglutinin HA1 polypeptide. In certain embodiments, an HA1N-terminal stem segment consists of amino acid residues correspondingapproximately to amino acids HA1_(N-term) through A_(p) of an HA1domain. HA1_(N-term) is the N-terminal amino acid of HA1 as recognizedby those of skill in the art. A_(p) is the cysteine residue in the HA1N-terminal stem segment that forms or is capable of forming a disulfidebond with a cysteine residue in an HA1 C-terminal stem segment. ResidueA_(p) is identified in influenza A hemagglutinin polypeptides in FIG. 1.Exemplary HA1 N-terminal stem segments are described herein. In certainembodiments, an HA1 N-terminal stem segment consists of amino acidresidues corresponding approximately to amino acids 1-52 of HA1 from anH3 hemagglutinin. Note that, in this numbering system, 1 refers to theN-terminal amino acid of the mature HA0 protein, from which the signalpeptide has been removed. Those of skill in the art will readily be ablerecognize the amino acid residues that correspond to the HA1 N-terminalstem segment of other influenza HA polypeptides, e.g., the amino acidresidues that correspond to the HA1 N-terminal stem segment of HA1 froman H1 hemagglutinin (see, e.g., FIG. 1).

“HA1 C-terminal stem segment” refers to a polypeptide segment thatcorresponds to the carboxy-terminal portion of the stem domain of aninfluenza hemagglutinin HA1 polypeptide. In certain embodiments, an HA1C-terminal stem segment consists of amino acid residues correspondingapproximately to amino acids A_(q) through HA1_(C-term) of an HA1domain. HA1_(C-term) is the C-terminal amino acid of the HA1 domain asrecognized by those of skill in the art. Residue A_(q) is identified ininfluenza A hemagglutinin polypeptides in FIG. 1. Exemplary HA1C-terminal stem segments are described herein. In certain embodiments,an HA1 C-terminal stem segment consists of amino acid residuescorresponding approximately to amino acids 277-346 of HA1 from an H3hemagglutinin. Note that, in this numbering system, 1 refers to theN-terminal amino acid of the mature HA0 protein, from which the signalpeptide has been removed. Those of skill in the art will readily be ablerecognize the amino acid residues that correspond to the HA1 C-terminalstem segment of other influenza HA polypeptides, e.g., the amino acidresidues that correspond to the HA1 C-terminal stem segment of HA1 froman H1 hemagglutinin (see, e.g., FIG. 1).

“HA2” refers to a polypeptide domain that corresponds to the HA2 domainof an influenza hemagglutinin polypeptide known to those of skill in theart. In certain embodiments, an HA2 consists of a stem domain, a luminaldomain, a transmembrane domain and a cytoplasmic domain (see, e.g.,Scheiffle et al., 2007, EMBO J. 16(18):5501-5508, the contents of whichare incorporated by reference in their entirety). In certainembodiments, an HA2 consists of a stem domain, a luminal domain and atransmembrane domain. In certain embodiments, an HA2 consists of a stemdomain and a luminal domain; in such embodiments, the HA2 might besoluble. In certain embodiments, an HA2 consists of a stem domain; insuch embodiments, the HA2 might be soluble.

As used herein, the term “heterologous” in the context of a polypeptide,nucleic acid or virus refers to a polypeptide, nucleic acid or virus,respectively, that is not normally found in nature or not normallyassociated in nature with a polypeptide, nucleic acid or virus ofinterest. For example, a “heterologous polypeptide” may refer to apolypeptide derived from a different virus, e.g., a different influenzastrain or subtype, or an unrelated virus or different species. Inspecific embodiments, when used in the context of a globular head domainof a chimeric influenza virus hemagglutinin described herein, the termheterologous refers to an influenza HA globular head domain that isassociated with an influenza HA stem domain that it would not normallybe found associated with (e.g., the head and stem domains of the HAwould not be found together in nature).

As used herein, the term “in combination,” in the context of theadministration of two or more therapies to a subject, refers to the useof more than one therapy (e.g., more than one prophylactic agent and/ortherapeutic agent). The use of the term “in combination” does notrestrict the order in which therapies are administered to a subject. Forexample, a first therapy (e.g., a first prophylactic or therapeuticagent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantlywith, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of asecond therapy to a subject.

As used herein, the term “infection” means the invasion by,multiplication and/or presence of a virus in a cell or a subject. In oneembodiment, an infection is an “active” infection, i.e., one in whichthe virus is replicating in a cell or a subject. Such an infection ischaracterized by the spread of the virus to other cells, tissues, and/ororgans, from the cells, tissues, and/or organs initially infected by thevirus. An infection may also be a latent infection, i.e., one in whichthe virus is not replicating.

As used herein, the term “influenza virus disease” refers to thepathological state resulting from the presence of an influenza virus(e.g., influenza A or B virus) in a cell or subject or the invasion of acell or subject by an influenza virus. In specific embodiments, the termrefers to a respiratory illness caused by an influenza virus.

As used herein, the phrases “IFN deficient system” or “IFN-deficientsubstrate” refer to systems, e.g., cells, cell lines and animals, suchas pigs, mice, chickens, turkeys, rabbits, rats, etc., which do notproduce IFN or produce low levels of IFN (i.e., a reduction in IFNexpression of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%,70-80%, 80-90% or more when compared to IFN-competent systems under thesame conditions), do not respond or respond less efficiently to IFN,and/or are deficient in the activity of one or more antiviral genesinduced by IFN.

As used herein, the term “like,” when used in the context of an“influenza-like virus,” refers an influenza virus that represents adifferent isolate of the referenced influenza virus, wherein the aminoacid sequence of said different isolate, or of the amino acid sequenceof the HA of said different isolate, is identical to, or nearlyidentical to, the amino acid sequence of the referenced influenza virusor the amino acid sequence of the HA of the referenced influenza virus;and/or the immune response against said different isolate confers fullprotection against the referenced influenza virus, and vice versa. Incertain embodiments, an influenza virus isolate that has an amino acidsequence that is nearly identical to the amino acid sequence of areferenced influenza virus has an amino acid sequence that is at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical tothe amino acid sequence of the referenced influenza virus. In certainembodiments, an influenza virus isolate that represents an“influenza-like virus” comprises an HA that has an amino acid sequencethat is nearly identical to the amino acid sequence of the HA of areferenced influenza virus, e.g., the HA of the influenza-like virus hasan amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of theHA of the referenced influenza virus.

As used herein, the numeric term “log” refers to log₁₀.

As used herein, the phrase “majority of the population is naive to,” inreference to a strain or subtype (e.g., an H1 subtype) of influenzavirus, refers to a strain or subtype of influenza virus that greaterthan 50% of the human population has presumably not been exposed to. Inspecific embodiments, the phrase “majority of the population is naiveto,” refers to a strain or subtype of influenza virus that at least 60%,70%, 75%, 80%, 85%, 90%, 95%, or 99% f the human population haspresumably not been exposed to.

As used herein, the phrase “multiplicity of infection” or “MOI” is theaverage number of infectious virus particles per infected cell. The MOIis determined by dividing the number of infectious virus particles added(ml added×PFU/ml) by the number of cells added (ml added×cells/ml).

As used herein, the term “nucleic acid” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid can be single-stranded or double-stranded.

As used herein, the term “polypeptide” refers to a polymer of aminoacids linked by amide bonds as is known to those of skill in the art. Asused herein, the term polypeptide can refer to a single polypeptidechain linked by covalent amide bonds. The term can also refer tomultiple polypeptide chains associated by non-covalent interactions suchas ionic contacts, hydrogen bonds, Van der Waals contacts andhydrophobic contacts. Those of skill in the art will recognize that theterm includes polypeptides that have been modified, for example bypost-translational processing such as signal peptide cleavage, disulfidebond formation, glycosylation (e.g., N-linked glycosylation), proteasecleavage and lipid modification (e.g. S-palmitoylation).

As used herein, the terms “prevent,” “preventing” and “prevention” inthe context of the administration of a therapy(ies) to a subject toprevent an influenza virus disease refer to one or more of theprophylactic/beneficial effects resulting from the administration of atherapy or a combination of therapies. In a specific embodiment, theterms “prevent,” “preventing” and “prevention” in the context of theadministration of a therapy(ies) to a subject to prevent an influenzavirus disease refer to one or more of the following effects resultingfrom the administration of a therapy or a combination of therapies: (i)the inhibition of the development or onset of an influenza virus diseaseor a symptom thereof; (ii) the inhibition of the recurrence of aninfluenza virus disease or a symptom associated therewith; and (iii) thereduction or inhibition in influenza virus infection and/or replication.

As used herein, the terms “purified” and “isolated” when used in thecontext of a polypeptide (including an antibody) that is obtained from anatural source, e.g., cells, refers to a polypeptide which issubstantially free of contaminating materials from the natural source,e.g., soil particles, minerals, chemicals from the environment, and/orcellular materials from the natural source, such as but not limited tocell debris, cell wall materials, membranes, organelles, the bulk of thenucleic acids, carbohydrates, proteins, and/or lipids present in cells.Thus, a polypeptide that is isolated includes preparations of apolypeptide having less than about 30%, 20%, 10%, 5%, 2%, or 1% (by dryweight) of cellular materials and/or contaminating materials. As usedherein, the terms “purified” and “isolated” when used in the context ofa polypeptide (including an antibody) that is chemically synthesizedrefers to a polypeptide which is substantially free of chemicalprecursors or other chemicals which are involved in the syntheses of thepolypeptide. In a specific embodiment, a chimeric influenzahemagglutinin (HA) polypeptide is chemically synthesized. In anotherspecific embodiment, an influenza hemagglutinin stem domain polypeptide,an influenza hemagglutinin head domain polypeptide, and/or a chimericinfluenza hemagglutinin polypeptide is isolated.

As used herein, the terms “replication,” “viral replication” and “virusreplication” in the context of a virus refer to one or more, or all, ofthe stages of a viral life cycle which result in the propagation ofvirus. The steps of a viral life cycle include, but are not limited to,virus attachment to the host cell surface, penetration or entry of thehost cell (e.g., through receptor mediated endocytosis or membranefusion), uncoating (the process whereby the viral capsid is removed anddegraded by viral enzymes or host enzymes thus releasing the viralgenomic nucleic acid), genome replication, synthesis of viral messengerRNA (mRNA), viral protein synthesis, and assembly of viralribonucleoprotein complexes for genome replication, assembly of virusparticles, post-translational modification of the viral proteins, andrelease from the host cell by lysis or budding and acquisition of aphospholipid envelope which contains embedded viral glycoproteins. Insome embodiments, the terms “replication,” “viral replication” and“virus replication” refer to the replication of the viral genome. Inother embodiments, the terms “replication,” “viral replication” and“virus replication” refer to the synthesis of viral proteins.

As used herein, the terms “stem domain polypeptide,” “HA stem domain,”“influenza virus hemagglutinin stem domain polypeptide” and “HA stalkdomain” refer to polypeptide comprising or consisting of one or morepolypeptide chains that make up a stem domain of an influenzahemagglutinin. A stem domain polypeptide might be a single polypeptidechain, two polypeptide chains or more polypeptide chains. Typically, astem domain polypeptide is a single polypeptide chain (i.e.corresponding to the stem domain of a hemagglutinin HA0 polypeptide) ortwo polypeptide chains (i.e. corresponding to the stem domain of ahemagglutinin HA1 polypeptide in association with a hemagglutinin HA2polypeptide). In specific embodiments, a stem domain polypeptide isderived from an influenza A H1 or H3 influenza virus hemagglutinin, oran influenza B influenza virus hemagglutinin.

As used herein, the terms “influenza virus hemagglutinin head domainpolypeptide,” “influenza virus hemagglutinin head domain,” “HA globularhead domain,” and “HA head domain” refer to the globular head domain ofan influenza hemagglutinin polypeptide. For example, for influenza Avirus, the globular head domain is generally understood to be presentbetween two key cysteine residues in the HA1 portion of the HA molecule.These cysteine residues are identified as “Ap” and “Aq” in FIG. 1 forvarious influenza A viruses.

As used herein, the terms “subject” or “patient” are usedinterchangeably to refer to an animal (e.g., birds, reptiles, andmammals). In a specific embodiment, a subject is a bird. In anotherembodiment, a subject is a mammal including a non-primate (e.g., acamel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, andmouse) and a primate (e.g., a monkey, chimpanzee, and a human). Incertain embodiments, a subject is a non-human animal. In someembodiments, a subject is a farm animal or pet. In another embodiment, asubject is a human. In another embodiment, a subject is a human infant.In another embodiment, a subject is a human child. In anotherembodiment, a subject is a human adult. In another embodiment, a subjectis an elderly human. In another embodiment, a subject is a prematurehuman infant.

As used herein, the term “premature human infant” refers to a humaninfant born at less than 37 weeks of gestational age.

As used herein, the term “human infant” refers to a newborn to 1 yearold human.

As used herein, the term “human child” refers to a human that is 1 yearto 18 years old.

As used herein, the term “human adult” refers to a human that is 18years or older.

As used herein, the term “elderly human” refers to a human 65 years orolder.

As used herein, the term “seasonal influenza virus strain” refers to astrain of influenza virus to which a subject population is exposed to ona seasonal basis. In specific embodiments, the term seasonal influenzavirus strain refers to a strain of influenza A virus. In specificembodiments, the term seasonal influenza virus strain refers to a strainof influenza virus that belongs to the H1 or the H3 subtype, i.e., thetwo subtypes that presently persist in the human subject population. Inother embodiments, the term seasonal influenza virus strain refers to astrain of influenza B virus.

As used herein, the terms “therapies” and “therapy” can refer to anyprotocol(s), method(s), compound(s), composition(s), formulation(s),and/or agent(s) that can be used in the prevention or treatment of aviral infection or a disease or symptom associated therewith. In certainembodiments, the terms “therapies” and “therapy” refer to biologicaltherapy, supportive therapy, and/or other therapies useful in treatmentor prevention of a viral infection or a disease or symptom associatedtherewith known to one of skill in the art. In some embodiments, theterm “therapy” refers to (i) a nucleic acid encoding a chimericinfluenza hemagglutinin (HA) polypeptide, (ii) a chimeric influenzahemagglutinin (HA) polypeptide, or (iii) a vector or compositioncomprising a nucleic acid encoding a chimeric influenza hemagglutinin(HA) polypeptide or comprising a chimeric influenza hemagglutinin (HA)polypeptide. In some embodiments, the term “therapy” refers to anantibody that specifically binds to a chimeric influenza virushemagglutinin polypeptide.

As used herein, the terms “treat,” “treatment,” and “treating” refer inthe context of administration of a therapy(ies) to a subject to treat aninfluenza virus disease or infection to obtain a beneficial ortherapeutic effect of a therapy or a combination of therapies. Inspecific embodiments, such terms refer to one, two, three, four, five ormore of the following effects resulting from the administration of atherapy or a combination of therapies: (i) the reduction or ameliorationof the severity of an influenza virus infection or a disease or asymptom associated therewith; (ii) the reduction in the duration of aninfluenza virus infection or a disease or a symptom associatedtherewith; (iii) the regression of an influenza virus infection or adisease or a symptom associated therewith; (iv) the reduction of thetiter of an influenza virus; (v) the reduction in organ failureassociated with an influenza virus infection or a disease associatedtherewith; (vi) the reduction in hospitalization of a subject; (vii) thereduction in hospitalization length; (viii) the increase in the survivalof a subject; (ix) the elimination of an influenza virus infection or adisease or symptom associated therewith; (x) the inhibition of theprogression of an influenza virus infection or a disease or a symptomassociated therewith; (xi) the prevention of the spread of an influenzavirus from a cell, tissue, organ or subject to another cell, tissue,organ or subject; (xii) the inhibition or reduction in the entry of aninfluenza virus into a host cell(s); (xiii) the inhibition or reductionin the replication of an influenza virus genome; (xiv) the inhibition orreduction in the synthesis of influenza virus proteins; (xv) theinhibition or reduction in the release of influenza virus particles froma host cell(s); and/or (xvi) the enhancement or improvement thetherapeutic effect of another therapy.

As used herein, in some embodiments, the phrase “wild-type” in thecontext of a virus refers to the types of a virus that are prevalent,circulating naturally and producing typical outbreaks of disease. Inother embodiments, the term “wild-type” in the context of a virus refersto a parental virus.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B, and FIG. 1C present a sequence alignment by CLUSTALWof representative sequences of 17 subtypes of influenza virus Ahemagglutinin (SEQ ID NOS:1-16 and 35, respectively). The residuedesignated A_(p) is the cysteine residue in the HA1 N-terminal stemsegment that forms or is capable of forming a disulfide bond with theresidue designated A_(q), a cysteine residue in an HA1 C-terminal stemsegment. The residue designated B_(q) represents the approximateN-terminal amino acid of the HA1 C-terminal short stem segmentsdescribed herein. The residue designated C_(q) represents theapproximate N-terminal amino acid of the HA1 C-terminal long stemsegments described herein. The residue designated C_(p) represents theapproximate C-terminal amino acid of the HA1 N-terminal long stemsegments described herein.

FIG. 2 provides a schematic of chimeric HAs with a conserved H1 stalkdomain and different globular head domains from distinct subtype HAs.

FIG. 3A and FIG. 3B. provide a novel influenza vaccine and diagnostictool platform to induce and analyze antibodies and reactive sera. A)Expression of chimeric HAs. Chimeric HAs consisting of the stalk domainof A/PR8/34 HA and the globular head domain of A/California/4/09(chimeric HA) as well as wild type HAs (PR8-HA and CAL09-HA) and a GFPcontrol were expressed in 293T cells. The upper Western blot was probedwith a PR8-specific antibody (PY102) whereas the blot on the lower sidewas probed with an antibody specific for Cal09 (39C2). B) Schematicdrawing of HA constructs expressed in A. The chimeric HA is composed ofthe A/PR/8/34 HA stalk domain and the 2009 A/California/04/09 globularhead domain.

FIG. 4A and FIG. 4B provide a schematic of chimeric HAs. A) Basicstructure of a chimeric HA. The globular head can be exchangedconveniently at disulfide bond Cys 52-Cys 277. B) Prime-boost regimewith sequential administration of chimeric HAs consisting of acompletely conserved stalk domain and a varying globular head domain.

FIG. 5 describes generation of a chimeric HA with the stalk of an H1 HAand the globular head of an H3 HA. A chimeric HA consisting of the stalkdomain of A/PR8/34 HA and the globular head domain of HK/68 (chimericH3) as well as wild type HAs (PR8-HA and HK68 HA) were expressed in 293Tcells. The upper Western blot was probed with a PR8-specific antibodywhereas the blot on the lower side was probed with an antibody specificfor H3.

FIG. 6 depicts a sequence comparison of the hemagglutinin proteinsequences of A/Hong Kong/1/1968 (H3), A/Perth/16/2009 (H3), A/PR/8/34(H1), A/Cal/4/09 (H1), A/Viet Nam/1203/04 (H5), andA/mallard/Alberta/24/01 (H7). The Cys52 and Cys277 amino acid residuesare specified (based on H3 numbering). The black shade indicatesconserved amino acids. The black wavy line represents the globular headregion of HAs. The starting points of HA1 and HA2 are indicated. Aminoacid sequences from the N-terminus to Cys52 of the HA of each of A/HongKong/1/1968 (H3), A/Perth/16/2009 (H3), A/PR/8/34 (H1), A/Cal/4/09 (H1),A/Viet Nam/1203/04 (H5), and A/mallard/Alberta/24/01 (H7) are presented,and correspond to SEQ ID NOs. 23-28, respectively. Amino acid sequencesfrom Cys277 of the HA of each of A/Hong Kong/1/1968 (H3),A/Perth/16/2009 (H3), A/PR/8/34 (H1), A/Cal/4/09 (H1), A/VietNam/1203/04 (H5), and A/mallard/Alberta/24/01 (H7) to the C-terminus arepresented, and correspond to SEQ ID NOs. 29-37, respectively.

FIG. 7A and FIG. 7B depict a schematic of chimeric hemagglutinins (A)Construction diagram of the chimeric PR8-cH1 HA. The chimeric HA wasconstructed by swapping the globular head domain located between Cys52and Cys277 of A/PR/8/34(H1) HA with that of the A/California/4/09(H1)HA. The resulting chimeric HA has the stalk region of A/PR8/34 (H1) HAwith a globular head domain of the A/California/4/09 (H1) HA designatedas PR8-cH1. (B) Schematic of the folded structures of the different wildtype and chimeric HAs, such as wild the type PR8 HA, the chimericPR8-cH1 HA, the chimeric PR8-cH5 HA, the wild type Perth HA, and thechimeric Perth-cH7 HA (from left to right). The full-length HAstructures were downloaded from the Protein Database (PDB): PR8 HA (PDBID 1RU7) and Perth HA (represented by HK68 HA, PDB ID 1MQN). Finalimages were generated with PyMol (Delano Scientific).

FIG. 8A and FIG. 8B depict the surface expression and functionalanalysis of chimeric HA constructs. (A) Surface expression of chimericHA constructs was evaluated in transiently transfected cells. At 24 hpost-transfection, 293T cells were trypsinized and cell surfaceexpression of chimeric HA proteins were analyzed by flow cytometry. Inthe upper panels, mock-transfected cells (left shaded region) arecompared to cells transfected with PR8 HA (right) or cells transfectedwith PR8-cH1 (right) or PR8-cH5 (right). In the bottom panels,mock-transfected cells (left shaded region) are compared to cellstransfected with Perth and Perth-cH7 constructs (right). (B)Luciferase-encoding pseudo-particles expressing chimeric HAs were usedto infect MDCK cells. The relative light units (RLU) generated in theluciferase assay indicate that pseudo-particles expressing chimeric HAswere able to enter the cells.

FIG. 9A and FIG. 9B describe the generation of recombinant virusesbearing chimeric hemagglutinins (A) Western blot analysis of therecombinant viruses. Extracts from MDCK cells mock infected or infectedwith the indicated viruses (16 hpi) at an MOI of 2 were prepared andprobed with antibodies: anti-A/PR8/HA(H1) (PY102), anti-A/Cal/09/HA(H1)(29C1), anti-A/VN/HA(H5) (M08), anti-H3/HA (12D1), anti-H7 (NR-3125),anti-A/NP (HT103) and anti-GAPDH as an internal loading control. (B)Immunofluorescence analysis of the MDCK cells infected with recombinantviruses using antibodies: anti-A/NP (HT103), anti-A/H1 HA (6F12),anti-A/PR8/HA (PY102), anti-A/Cal/09/HA (29C1), anti-A/VN/HA (M08),anti-H3/HA (12D1), and anti-A/H7 virus (NR-3152).

FIG. 10A and FIG. 10B describe the growth kinetics and plaque phenotypesof recombinant viruses. (A) 10-day old embryonated chicken eggs wereinfected with wild-type or recombinant virus with 100 pfu per egg andviral growth monitored for 72 hours post infection. (B) The plaquephenotype of recombinant viruses was assessed by plaque assay. MDCKcells were infected with either a wild-type or recombinant virus and at48 hours post infection immuno-stained to reveal plaque phenotype usingthe antibody against A/NP (HT103).

FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, and FIG. 11F depict animmunofluorescence analysis of cells transfected with chimeric H6hemagglutinin. 293T cells were transfected with 1 μg of pCAGGS plasmidexpressing chimeric H6 hemagglutinin. Sera from animals that receivedDNA (A), Cal/09 infection (B), DNA and Cal/09 infection (C), or Cal/09split vaccine (D) were added to transfected cells and visualized byfluorescence microscopy following incubation with an Alexa Fluor594-conjugated anti-mouse IgG. As controls, the cross-reactive H1 stemantibody C179 (E) or the antibody PY102 (F) directed against theglobular head of PR8 were added to transfected cells and visualized byfluorescence microscopy following incubation with an Alexa Fluor594-conjugated anti-mouse IgG.

FIG. 12 demonstrates that DNA prime and chimeric virus boost conferprotection to animals challenged with lethal influenza virus challenge.Animals were either treated with DNA alone, chimeric H9 virus alone, DNAprime and chimeric H9 virus boost, or with inactivated PR8 virus. Micewere then challenged with 5×10⁴ PFU of PR8 virus, instilledintranasally, and the weight of the animals was monitored for 14 days.

FIG. 13 demonstrates reactivity of stalk specific antibodies to cH6protein as determined by ELISA.

FIG. 14A and FIG. 14B depict a schematic representation of chimeric HA(cHA) proteins (A) and cHA expression in MDCK cells (B). Chimeric HA(cHA) proteins and recombinant chimeric virus. (A) Schematicrepresentation of cHAs. The globular head domain is defined as theintervening amino acid sequence between residues C52 and C277 (H3numbering). Using this disulfide bond as the demarcation between headand stalk, exotic HA heads were introduced atop heterologous stalks. Thestalk domain is defined as the remaining portions of HA1 and HA2subunits. CT, cytoplasmic tail; SP, signal peptide; TM, transmembranedomain. The full-length HA structures were downloaded from the ProteinDatabase (PDB): PR8 (H1) HA (PDB ID 1RU7) and A/guinea fowl/HongKong/WF10/99 HA [represented by A/swine/Hong Kong/9/98 (H9; PDB ID1JSD)]. Final images were generated with PyMol (Delano Scientific).Because no structure of an H6 HA has been published, the image of thehead-folding of the PR8 HA is used for the cH6/1 construct. (B)Immunofluorescence to confirm expression of cHA. MDCK cells wereinfected with either WT PR8 or cH9/1 N3 virus, or they weremock-infected. Antibodies specific for the head and stalk of PR8 virusas well as an antibody with H9 reactivity were used to confirm cHAexpression. (Magnification bar: 40×).

FIG. 15A, FIG. 15B, FIG. 15C, FIG. 15D, and FIG. 15E show that adultpatients infected with pandemic H1N1 virus have high titers ofneutralizing antibodies that are reactive with the HA stalk. Reactivityof sera of pH1N1-infected adults (n=9), children not infected with pH1N1(n=5), and adults not infected with pH1N1 virus (n=11) with cH6/1protein (A), cH9/1 protein; (B), the LAH of the HA2 protein (anti-LAHantibody was used as a positive control; (C), H5 HA protein (mousepolyclonal serum raised against H5 HA was used as a positive control anda pan-H3 antibody, 12D1, was used as negative control; (D) (13), or H3HA protein (12D1 was used as a positive control and mouse polyclonalserum raised against H5 HA was used as a negative control; (E). All wereassessed by ELISA; data points represent average titers with SE orreactivity of pooled samples.

FIG. 16A, FIG. 16B, and FIG. 16C show that adult patients infected withpandemic H1N1 virus have high titers of neutralizing antibodies that arespecific for the HA stalk (A and B). Sera from pH1N1-infected (n=14) andadults not infected with pH1N1 (n=5) were pooled separately, and totalIgG from both pools was purified. Neutralizing capability of stalkantibodies was assessed by plaque reduction assay using cH9/1 N3 virus.Data points represent the mean and SE of two experiments. Plaques wereimmunostained with anti-H9 antibody G1-26. (B) shows plaque reduction ofthe four dilutions of sera shown along the top. (C) Pseudotype particleneutralization assay measures neutralizing antibody activity of thehuman-purified IgG preparations (sera from pH1N1-infected adults andadults not infected with pH1N1). Total IgG concentrations were 50, 10,and 2 μg/mL. As a positive control, the stalk-specific monoclonalantibody 6F12 was used.

FIG. 17A, FIG. 17B, and FIG. 17C show expression and function of cH6/1and cH9/1 protein. A) Coomasie gel of 2 μg cH6/1 and cH9/1 protein. M,marker proteins. (B) Western blot analysis of baculovirus expressed cHAproteins. Lane 1, cH6/1 protein; lane 2, cH9/1 protein; lane 3, WT PR8HA; lane 4, WT H3 HA. Blots were probed with antibodies known to reactwith the stalk of PR8 virus (rabbit polyclonal anti-HA2) or H3 viruses(mouse mAb 12D1) and the globular head of H6 (goat polyclonal anti-H6)or H9 viruses (mouse mAb G1-26) to confirm the identity of baculovirusexpressed cHAs. mAb 12D1 reacts with both HA0 and HA2 (H3 proteinpreparation is cleaved, resulting in two distinct bands). (C) Plaqueassay of cH9/1 N3 reassortant virus. Reassortant cH9/1 N3 virus plaquephenotype is similar to plaques made by WT PR8 virus. Plaques wereimmunostained with PY102 and anti-H9 antibody G1-26.

FIG. 18A, FIG. 18B, FIG. 18C, and FIG. 18D show that monoclonalantibodies directed against the stalk of influenza virus HA bind andneutralize cHAs. (A) Stalk antibody C179 was used to test reactivity tocH6/1 baculovirus-expressed protein by ELISA. C179 reacted with cH9/1 ina dose-dependent manner. (B) Stalk antibody C179 was used to testreactivity to cH9/1 baculovirus-expressed protein by ELISA. C179 reactedwith cH9/1 in a dose-dependent manner (C and D). Antibody 6F12neutralizes cH9/1 N3 virus replication. 6F12 was used to assess theability of stalk-specific monoclonal antibodies to neutralize cH9/1 N3virus by plaque reduction assay. D shows plaque reduction of cH9/1 N3virus using five dilutions of mAb 6F12 (100, 20, 4, 0.8, and 0.16μg/mL). Plaques were immunostained with anti-H9 antibody G1-26.

FIG. 19A and FIG. 19B depict schematics of chimeric hemagglutinins FIG.19A shows a diagram of wild-type and cH1/1 viruses. The chimeric HA wasconstructed by swapping the globular head domain located between Cys52and Cys277 of PR8 (H1) HA with that of the A/California/4/09 (H1) HA.The resulting chimeric HA has the stalk region of A/PR8/34 (H1) HA witha globular head domain of the A/California/4/09 (H1) HA and isdesignated as cH1/1. FIG. 19B shows theoretical schematics of the foldedstructures of the different wild type and chimeric HAs. From left toright: wild type PR8 HA, the chimeric cH1/1 HA, the chimeric cH5/1 HA,the wild type Perth HA, the chimeric cH7/3 HA, and the chimeric cH5/3HA.

FIG. 20 depicts a table comparing amino acid identity between H1, H3, H5and H7 HAs used in this study. Percent amino acid identity wascalculated using ClustalW (excluding the signal peptide). Percent aminoacid identity is compared for full length HA, as well as the globularhead and stalk domains. Grey bars indicate 100% identity.

FIG. 21 shows the surface expression of chimeric HA constructs. Surfaceexpression of chimeric HA constructs was evaluated in transientlytransfected or infected cells. At 48 h post-transfection, 293T cellswere trypsinized and cell surface expression of chimeric HA proteinswere analyzed by flow cytometry. In the upper panels, mock-transfectedcells (grey) are compared to cells transfected with PR8 HA (black line)or cells transfected with cH1/1 (black line) or cH5/1 (black line). Inthe center panels, mock-transfected cells (grey) are compared to cellstransfected with Perth/09, cH7/3 (black line) and cH5/3 constructs(black line). In the bottom panels, MDCK cells were infected withPerth/09, cH7/3 and cH5/3 expressing recombinant viruses. At 12 hpost-infection the cell surface expression of the different HAs wereanalyzed using flow cytometry.

FIG. 22 demonstrates the ability of the chimeric HAs to enter MDCKcells. Luciferase-encoding pseudoparticles expressing chimeric HAs wereused to transduce MDCK cells. The relative light units (RLU) generatedin the luciferase assay indicates that pseudoparticles expressingchimeric HAs are able to enter cells.

FIG. 23 shows a Western blot analysis of cells infected with therecombinant cHA-expressing viruses. Extracts from MDCK cells mockinfected or infected with the indicated viruses at an MOI of 2 wereprepared and probed with antibodies at 16 hpi: anti-A/PR8/HA (H1)(PY102), anti-A/Cal/09/HA (H1) (29E3), anti-A/VN/HA (H5) (mAb #8),anti-H3/HA (12D1), anti-H7 (NR-3152), anti-A/NP (HT103) and anti-GAPDHas an loading control.

FIG. 24 depicts an immunofluorescence analysis of MDCK cells infectedwith recombinant viruses using antibodies: anti-A/NP (HT103), anti-A/H1HA (6F12), anti-A/PR8/HA (PY102), anti-A/Cal/09/HA (29E3), anti-A/VN/HA(mAb #8), anti-H3/HA (12D1), and anti-A/H7 virus (NR-3152).

FIG. 25A and FIG. 25B depict the growth kinetics and plaque phenotypesof wild type and recombinant viruses. (A) 10-day old embryonated chickeneggs were infected with 100 pfu per egg of wild-type or recombinantvirus and viral growth was monitored for 72 hours post infection. Datapoints represent the average and standard deviation of experimentalreplicates. (B) The plaque phenotypes of recombinant viruses wereassessed by plaque assay. MDCK cells were infected with either awild-type or recombinant virus. Cells were fixed 48 hours post infectionand immunostained to reveal plaque phenotypes using the antibody againstA/NP (HT103).

FIG. 26A and FIG. 26B depict that stalk-specific monoclonal antibodyneutralizes cHA-expressing viruses and pseudotype particles. The abilityof a mAb (KB2) to neutralize cHA-expressing viruses or pseudotypeparticles was assessed by plaque reduction assay (A) or pseudotypeparticle inhibition assay (B). MDCK cells were infected or transducewith cHA-expressing viruses or pseudotype particles in the presence ofthe indicated amount (ug/mL) of the mAb or without antibody. Plaqueformation or luciferase activity was used as a readout to determine thedegree of inhibition by the mAb. (A) The mAb neutralizes cH1/1 (blackboxes) and cH5/1 (black triangles) virus replication in a dose dependentmanner, with 100% inhibition at concentrations above 100 ug/mL. Datapoints represent the average and standard deviation of experimentalreplicates. (B) The mAb also inhibits entry of cH1/1 (black boxes)pseudotype particles in a dose dependent manner, with completeinhibition above 4 ug/mL. Data points represent the average and standarddeviation of experimental replicates. The pseudotype inhibition assayswere processed independently.

FIG. 27A, FIG. 27B, and FIG. 27C demonstrate that sequential vaccinationwith cHA constructs elicits HA stalk-specific antibodies and providesprotection from lethal challenge. Mice were primed with 20 μg of cH9/1protein, administered with adjuvant intranasally and intraperitoneally.Three weeks later, mice were boosted with 20 μg cH6/1 protein,administered with adjuvant intranasally and intraperitoneally. Ascontrols, mice were primed and boosted in a similar fashion using BSA,or given inactivated FM1 virus intramuscularly. Animals were bled andchallenged three weeks after the last vaccination with 5LD50 of A/FortMonmouth/1/1947 (FM1) virus. (A) ELISA plates were coated with cH5/1 N1virus in order to assess the degree of stalk reactivity elicited throughvaccination (BSA+BSA is the bottom line of the graph). (B) Mice wereweighed daily for 14 days to assess protection from challenge. (C)Kaplan Meier curve depicting survival rate post challenge. cH9/1+cH6/1vaccinated mice had a statistically higher survival rate compared to BSAcontrols (p=0.0003)

FIG. 28A, FIG. 28B, FIG. 28C, FIG. 28D, and FIG. 28E demonstrate thatvaccination with cH6/1 elicits stalk-specific immunity that mediatesprotection from cH9/1 N1 viral challenge. Animals were inoculated withYAM-HA virus in order to simulate prior infection with or vaccinationagainst influenza virus. Three weeks later, animals were vaccinated withcH6/1 or BSA with adjuvant, intranasally and intraperitoneally. Controlanimals were inoculated with wild-type B/Yamagata/16/1988 and vaccinatedin a similar fashion with BSA, or given inactivated cH9/1 N1 virusintramuscularly. Animals were bled and challenged with 250LD₅₀ cH9/1 N1virus three weeks after vaccination. (A) Animals were weighed for 8 daysin order to assess protection from challenge. On days 3-5, YAM-HA+cH6/1animals demonstrated statistically less weight loss compared to theYAM-HA+BSA cohort (p<0.05). (B) Kaplan Meier curve depicting survival.Statistically different survival rates were seen in the YAM-HA+cH6/1group compared to the YAM-HA+BSA cohort (p=0.038), as well as naïve andWT YAM+BSA animals (p<0.0001). Survival rate of YAM-HA+BSA cohort wasnot statistically different from that of the WT-YAM+BSA cohort(p=0.058). (C) ELISA plates were coated with cH5/1 N1 virus in order toassess the degree of stalk reactivity elicited through vaccination. (D)Results of a plaque reduction assay using the cH5/1 virus are depicted.(E) Animals were vaccinated, bled and total IgG was harvested forH5-based pseudoparticle entry inhibition assay. Percent inhibition wasassessed as a decrease in luciferase expression compared to controls.

FIG. 29A, FIG. 29B, and FIG. 29C demonstrate that vaccination with cH6/1protects mice from lethal H5 influenza virus challenge. Animals wereinoculated with YAM-HA virus in order to simulate prior infection withor vaccination against influenza virus. Three weeks later, animals werevaccinated with cH6/1 or BSA with adjuvant, intranasally andintraperitoneally. Control animals were inoculated with wild-typeB/Yamagata/16/1988 and vaccinated in a similar fashion with BSA or giveninactivated cH5/1 N1 virus intramuscularly. Animals were bled andchallenged with 10LD₅₀ of the 2:6 H5 reassortant in the PR8 background(see, e.g., Steel et al., 2009, J Virol 83:1742-1753). (A) Kaplan Meiercurve depicting survival. Differences in survival rates approachedstatistical significance when comparing the YAM-HA+cH6/1 group to theYAM-HA+BSA cohort (p=0.06). (B) Length of survival was on average longerin animals inoculated with YAM-HA and vaccinated with cH6/1 than animalsvaccinated with BSA (p=0.037), as well as naïve and WT YAM/BSA controls(p<0.001). (C) ELISA plates were coated with cH5/1 N1 virus in order toassess the degree of stalk reactivity elicited through vaccination. 1:50serum dilutions were plotted against % maximum weight loss. One valuewas determined to be an outlier and was omitted from analysis. Forlinear regression, R²=0.56 and p=0.02.

FIG. 30A, FIG. 30B, FIG. 30C, FIG. 30D, FIG. 30E, FIG. 30F, FIG. 30G,FIG. 30H, FIG. 30I, and FIG. 30J demonstrate that vaccination with cHAelicits stalk-specific immunity that mediates protection from H1N1 viruschallenge. (A-F) Animals were primed with DNA encoding cH9/1 and thenwere vaccinated with cH6/1 and boosted with cH5/1 soluble protein (n=10)or BSA (n=5), while positive control mice received inactivated virusintramuscularly (n=5). (A) Animals were vaccinated and challenged withFM1 virus; mice were weighed daily, and weight loss over time is shownas change in percentage of initial weight. (B) Graph depicting survivalof challenged mice in (A). (C) Animals were vaccinated and challengedwith pH1N1 virus; mice were weighed daily, and weight loss over time isshown as change in percentage of initial weight. (D) Graph depictingsurvival of challenged mice in (C). (E) Animals were vaccinated andchallenged with PR8 virus; mice were weighed daily, and weight loss overtime is shown as change in percentage of initial weight. (F) Graphdepicting survival of challenged mice in (E). (G) Reactivity to H1 HA ofserum from animals vaccinated as described above in A-F and below in H-Iand challenged with 5 LD₅₀ of A/FM/1/1947 (A, B), 10 LD₅₀ ofA/Netherland/602/2009 (C, D), or 5 LD₅₀ of A/PR/8/1934 (E, F, H, I). (H)Animals were vaccinated as described above in A-F (square, n=4) or werenaive (triangle, n=3), while positive control mice received inactivatedPR8 virus intramuscularly (X mark, n=5). CD8 T cells were depleted priorto challenge with PR8 virus. Mice were weighed daily, and weight lossover time is shown as change in percentage of initial weight. (I) Graphdepicting survival of challenged mice in (H). (J) Animals werevaccinated as described for A-F. Total IgG was purified for use inH2-based pseudoparticle entry inhibition assay. Percent inhibition wasassessed as a decrease in luciferase expression compared to controls.Fab fragment CR6261 was used as a positive control.

FIG. 31A, FIG. 31B, and FIG. 31C. Schematic of wild type HA andexpression constructs. (A) Uncleaved full length influenza virushemagglutinin. The signal peptide is the leftmost component, the HAectodomain is the middle component and the transmembrane- and endodomainare the rightmost component. (B) Expression construct with trimerizationdomain. The transmembrane- and endodomain was swapped with a thrombincleavage site (third component from left), a T4 trimerization domain(fourth component from left) and a hexahistidine tag (6×his tag, fifthcomponent from left) at position V503 (H3 numbering). (C) Expressionconstruct without trimerization domain. The transmembrane- andendodomain was swapped with a hexahistidine tag (6×his tag, rightmostcomponent) at amino acid position 509 (H1, H2 and H5) or 508 (H3)respectively (H3 numbering).

FIG. 32A, FIG. 32B, and FIG. 32C. Introduction of a trimerization domaininfluences stability and formation of oligomers in recombinant HAs. (A)Analysis of recombinant HAs with and without trimerization domain byreducing, denaturing SDS-PAGE. Recombinant HAs that are expressed withtrimerization domain (+) show higher stability than HAs expressedwithout (−). Uncleaved HA (HA0) and cleavage products (HA1/degr.product; HA2) are indicated by arrows. (B) Reducing, denaturing SDS-PAGEanalysis of crosslinked HAs. Different species of HA are indicated inthe blot. High molecular multimers are indicated by H, trimers by T,dimers by D and monomers by M. (C) Left panel (boxes 1-4): Western blotanalysis of reduced, denatured and cross-linked group 1 HAs from Bprobed with a anti-hexahistidine-tag antibody. Right panel (rightmostbox): Cross-linking control (IgG) with BS³ analyzed on a SDS-PAGE.Different species (full antibody, heavy chain, light chain) areindicated by arrows. Molecular weights of the marker bands are indicatedon the left of each panel.

FIG. 33A and FIG. 33B. Binding of stalk-reactive antibodies torecombinant PR8 (H1) and Cal09 (H1) HAs. (A) Binding of stalk-reactiveantibodies C179, CR6261 and 6F12 and head-reactive antibody PY102 andPR8 antiserum to recombinant soluble PR8 HA without (w/o T4 trim.domain, triangle lines) or with (w/ T4 trim. domain, boxed lines)trimerization domain. (B) Binding of stalk-reactive antibodies C179,CR6261 and 6F12 and head-reactive antibody 7B2 and Cal09 antiserum torecombinant soluble Cal09 HA without (w/o T4 trim. domain, trianglelines) or with (w/ T4 trim. domain, boxed lines) trimerization domain.

FIG. 34A and FIG. 34B. Binding of stalk-reactive antibodies torecombinant JAP57 (H2) and VN04 (H5) HAs. (A) Binding of stalk-reactiveantibodies C179 and CR6261 and head-reactive antibody 8F8 and H2antiserum to recombinant soluble JAP57 HA without (w/o T4 trim. domain,triangle lines) or with (w/ T4 trim. domain, boxed lines) trimerizationdomain. (B) Binding of stalk-reactive antibodies C179 and CR6261 andhead-reactive antibody mAb#8 and H5 antiserum to recombinant solubleVN04 HA without (w/o T4 trim. domain, triangle lines) or with (w/ T4trim. domain, boxed lines) trimerization domain.

FIG. 35A and FIG. 35B. Binding of stalk-reactive antibodies to group 2HAs. (A) Binding of stalk-reactive antibodies 12D1 and CR8020 andhead-reactive antibody XY102 and H3 antiserum to recombinant solubleHK68 HA without (w/o T4 trim. domain, triangle lines) or with (w/ T4trim. domain, boxed lines) trimerization domain. (B) Binding ofstalk-reactive antibodies 12D1 and CR8020 and H3 antiserum torecombinant soluble Wisc05 HA without (w/o T4 trim. domain, trianglelines) or with (w/ T4 trim. domain, boxed lines) trimerization domain.

FIG. 36 depicts an exemplary chimeric influenza virus hemagglutininpolypeptide (SEQ ID NO: 21) comprising the stem domain of an influenza Bvirus (B/Florida/4/2006) and the globular head domain of an influenza Avirus of the H5 subtype (A/Vietnam/1203/2004).

5. DETAILED DESCRIPTION

In one aspect, provided herein are chimeric influenza hemagglutinin (HA)polypeptides that induce a cross-protective immune response against theconserved HA stem domain of influenza viruses. The chimeric influenza HApolypeptides provided herein comprise a stable HA stem domain and aglobular HA head domain that is heterologous to the stem domain (i.e.the head and stem domains are derived from different strains and/orsubtypes of influenza virus).

In another aspect, provided herein are compositions comprising one ormore of the chimeric influenza hemagglutinin polypeptides describedherein (e.g., compositions comprising soluble chimeric influenzahemagglutinin polypeptides described herein, viruses comprising thechimeric influenza hemagglutinin polypeptides described herein, virusescomprising genomes engineered to encode the chimeric influenzahemagglutinin polypeptides described herein, expression vectorscomprising the chimeric influenza hemagglutinin polypeptides describedherein, expression vectors comprising genomes engineered to encode thechimeric influenza hemagglutinin polypeptides described herein, nucleicacids encoding the chimeric influenza hemagglutinin polypeptidesdescribed herein, etc.).

In another aspect, provided herein are vaccine formulations comprisingone or more of the chimeric influenza hemagglutinin polypeptidesdescribed herein. In a specific embodiment, provided herein is amonovalent vaccine comprising one of the chimeric influenzahemagglutinin polypeptides described herein. In another specificembodiment, provided herein is a bivalent vaccine comprising two of thechimeric influenza hemagglutinin polypeptides described herein (i.e.,two distinct chimeric influenza hemagglutinin polypeptides). In anotherspecific embodiment, provided herein is a trivalent vaccine comprisingthree of the chimeric influenza hemagglutinin polypeptides describedherein (i.e., three distinct chimeric influenza hemagglutininpolypeptides). The vaccine formulations provided herein may comprise,for example, subunit vaccines comprising one or more of the chimericinfluenza hemagglutinin polypeptides described herein (e.g.,compositions comprising chimeric influenza hemagglutinin polypeptides,e.g., soluble chimeric influenza hemagglutinin polypeptides); liveinfluenza viruses (e.g., live attenuated influenza viruses) that expressone or more of the chimeric influenza hemagglutinin polypeptidesdescribed herein; live influenza viruses (e.g., live attenuatedinfluenza viruses) comprising a genome that encodes one or more of thechimeric influenza hemagglutinin polypeptides described herein; killedinfluenza viruses that express one or more of the chimeric influenzahemagglutinin polypeptides described herein; killed influenza virusescomprising a genome that encodes one or more of the chimeric influenzahemagglutinin polypeptides described herein; virus/viral-like particles(“VLPs”) that contain one or more of the chimeric influenzahemagglutinin polypeptides described herein; split virus vaccines,wherein said virus expresses one or more of the chimeric influenzahemagglutinin polypeptides described herein and/or comprises a genomethat encodes one or more of the chimeric influenza hemagglutininpolypeptides described herein; viral expression vectors (e.g.,non-influenza virus expression vectors) that express one or more of thechimeric influenza hemagglutinin polypeptides described herein; andbacterial expression vectors that express one or more of the chimericinfluenza hemagglutinin polypeptides described herein.

The vaccine formulations described herein can elicit highly potent andbroadly neutralizing antibodies against the HA stem domain of thechimeric influenza hemagglutinin polypeptides. Such “universal” vaccinescan be used to induce and/or boost cross-protective immune responsesacross influenza virus subtypes.

In another aspect, provided herein are methods of immunizing a subjectagainst an influenza virus disease or infection comprising administeringto the subject a composition comprising one or more of the chimericinfluenza hemagglutinin (HA) polypeptides described herein or a vaccineformulation described herein.

In another aspect, provided herein are kits comprising one or more ofthe chimeric influenza hemagglutinin (HA) polypeptides described hereinor a vaccine formulation described herein. The kits provided herein mayfurther comprise one or more additional components, e.g., an antibodythat specifically binds one or more of the chimeric influenzahemagglutinin (HA) polypeptides provided in the kit.

5.1 Chimeric Influenza Virus Hemagglutinin Polypeptides

Provided herein are chimeric influenza virus hemagglutinin polypeptidescomprising or consisting of an influenza virus hemagglutinin globularhead domain polypeptide and an influenza virus hemagglutinin stem domainpolypeptide, wherein said influenza virus hemagglutinin head domainpolypeptide is heterologous to said influenza virus hemagglutinin stemdomain polypeptide (e.g., the influenza virus hemagglutinin globularhead domain polypeptide and the influenza virus hemagglutinin stemdomain polypeptide are derived from different influenza virushemagglutinin subtypes).

A full-length influenza hemagglutinin typically comprises an HA1 domainand an HA2 domain. The stem domain is formed by two segments of the HA1domain and most or all of the HA2 domain. The two segments of the HA1domain are separated, in primary sequence, by the globular head domain(see, e.g., the amino acid residues between the residues designatedA_(p) and A_(q) in FIG. 1). In certain embodiments, the chimericinfluenza virus hemagglutinin polypeptides described herein maintainsuch a structure. That is, in certain embodiments, the chimericinfluenza virus hemagglutinin polypeptides described herein comprise astable stem structure composed of an HA1 domain and an HA2 domain, and aglobular head domain separating the two segments of the HA1 domain (inprimary sequence), wherein said globular head domain is heterologous tothe stem domain formed by the other segments of the HA1 domain and theHA2 domain.

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza virus of the H1 subtype and (ii) theglobular head domain of the hemagglutinin from an influenza virus of theH5 subtype (sometimes referred to herein as a “cH5/1 chimeric influenzahemagglutinin polypeptide”). In a specific embodiment, the stem domainof a cH5/1 chimeric influenza hemagglutinin polypeptide is the stemdomain of A/California/4/2009 (H1N1) HA (or the stem domain of anA/California/4/2009-like influenza virus HA). In another specificembodiment, the stem domain of a cH5/1 chimeric influenza hemagglutininpolypeptide is the stem domain of A/California/4/2009 (H1N1) HA (or thestem domain of an A/California/4/2009-like influenza virus HA) and theglobular head domain of the cH5/1 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Vietnam/1203/2004 (H5) HA.In another specific embodiment, the stem domain of a cH5/1 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/California/4/2009 (H1N1) HA (or the stem domain of anA/California/4/2009-like influenza virus HA) and the globular headdomain of the cH5/1 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Indonesia/5/2005 (H5) HA. In another specificembodiment, the stem domain of a cH5/1 chimeric influenza hemagglutininpolypeptide is the stem domain of A/California/4/2009 (H1N1) HA (or thestem domain of an A/California/4/2009-like influenza virus HA) and theglobular head domain of the cH5/1 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Anhui/1/2005 (H5) HA. Inanother specific embodiment, the stem domain of a cH5/1 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/California/4/2009 (H1N1) HA (or the stem domain of anA/California/4/2009-like influenza virus HA) and the globular headdomain of the cH5/1 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA. Inanother specific embodiment, the stem domain of a cH5/1 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/California/4/2009 (H1N1) HA (or the stem domain of anA/California/4/2009-like influenza virus HA) and the globular headdomain of the cH5/1 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/turkey/Turkey/1/2005 (H5) HA. In anotherspecific embodiment, the stem domain of a cH5/1 chimeric influenzahemagglutinin polypeptide is the stem domain of A/California/4/2009(H1N1) HA (or the stem domain of an A/California/4/2009-like influenzavirus HA) and the globular head domain of the cH5/1 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Whooperswan/Mongolia/244/2005 (H5) HA.

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza virus of the H3 subtype and (ii) theglobular head domain of the hemagglutinin from an influenza virus of theH5 subtype (sometimes referred to herein as a “cH5/3 chimeric influenzahemagglutinin polypeptide”). In a specific embodiment, the stem domainof a cH5/3 chimeric influenza hemagglutinin polypeptide is the stemdomain of A/Victoria/361/2011 (H3N2) HA. In another specific embodiment,the stem domain of a cH5/3 chimeric influenza hemagglutinin polypeptideis the stem domain of A/Victoria/361/2011 (H3N2) HA and the globularhead domain of the cH5/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Vietnam/1203/2004 (H5) HA. In anotherspecific embodiment, the stem domain of a cH5/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/Victoria/361/2011(H3N2) HA and the globular head domain of the cH5/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Indonesia/5/2005 (H5) HA. In another specific embodiment, the stemdomain of a cH5/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Victoria/361/2011 (H3N2) HA and the globular headdomain of the cH5/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Anhui/1/2005 (H5) HA. In another specificembodiment, the stem domain of a cH5/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Victoria/361/2011 (H3N2) HA and theglobular head domain of the cH5/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Bar headedgoose/Quinghai/1A/2005 (H5) HA. In another specific embodiment, the stemdomain of a cH5/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Victoria/361/2011 (H3N2) HA and the globular headdomain of the cH5/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/turkey/Turkey/1/2005 (H5) HA. In anotherspecific embodiment, the stem domain of a cH5/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/Victoria/361/2011(H3N2) HA and the globular head domain of the cH5/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Whooperswan/Mongolia/244/2005 (H5) HA.

In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA. In another specific embodiment, thestem domain of a cH5/3 chimeric influenza hemagglutinin polypeptide isthe stem domain of A/harbor seal/Massachusetts/1/2011 (H3N8) HA and theglobular head domain of the cH5/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Vietnam/1203/2004 (H5) HA.In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA and the globular head domain of thecH5/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Indonesia/5/2005 (H5) HA. In another specific embodiment,the stem domain of a cH5/3 chimeric influenza hemagglutinin polypeptideis the stem domain of A/harbor seal/Massachusetts/1/2011 (H3N8) HA andthe globular head domain of the cH5/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Anhui/1/2005 (H5) HA. Inanother specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA and the globular head domain of thecH5/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA. In anotherspecific embodiment, the stem domain of a cH5/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA and the globular head domain of thecH5/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/turkey/Turkey/1/2005 (H5) HA. In another specificembodiment, the stem domain of a cH5/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/harbor seal/Massachusetts/1/2011(H3N8) HA and the globular head domain of the cH5/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/Whooperswan/Mongolia/244/2005 (H5) HA.

In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA. In another specific embodiment, the stemdomain of a cH5/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Indiana/10/2011 (H3N2) HA and the globular head domainof the cH5/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Vietnam/1203/2004 (H5) HA. In another specificembodiment, the stem domain of a cH5/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Indiana/10/2011 (H3N2) HA and theglobular head domain of the cH5/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Indonesia/5/2005 (H5) HA.In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA and the globular head domain of the cH5/3chimeric influenza hemagglutinin polypeptide is the globular head domainof A/Anhui/1/2005 (H5) HA. In another specific embodiment, the stemdomain of a cH5/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Indiana/10/2011 (H3N2) HA and the globular head domainof the cH5/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA. Inanother specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA and the globular head domain of the cH5/3chimeric influenza hemagglutinin polypeptide is the globular head domainof A/turkey/Turkey/1/2005 (H5) HA. In another specific embodiment, thestem domain of a cH5/3 chimeric influenza hemagglutinin polypeptide isthe stem domain of A/Indiana/10/2011 (H3N2) HA and the globular headdomain of the cH5/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Whooperswan/Mongolia/244/2005 (H5) HA.

In a specific embodiment, a cH5/3 chimeric influenza hemagglutininpolypeptide provided herein does not comprise the globular head domainof A/Vietnam/1203/2004 (H5) HA. In another specific embodiment, a cH5/3chimeric influenza hemagglutinin polypeptide does not comprise the stemdomain of A/Perth/16/2009 (H3) HA.

In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA. In another specific embodiment, the stemdomain of a cH5/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Perth/16/2009 (H3N2) HA and the globular head domain ofthe cH5/3 chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/Vietnam/1203/2004 (H5) HA. In another specificembodiment, the stem domain of a cH5/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Perth/16/2009 (H3N2) HA and theglobular head domain of the cH5/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Indonesia/5/2005 (H5) HA.In another specific embodiment, the stem domain of a cH5/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA and the globular head domain of the cH5/3chimeric influenza hemagglutinin polypeptide is the globular head domainof A/Anhui/1/2005 (H5) HA. In another specific embodiment, the stemdomain of a cH5/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Perth/16/2009 (H3N2) HA and the globular head domain ofthe cH5/3 chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA. In anotherspecific embodiment, the stem domain of a cH5/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/Perth/16/2009 (H3N2)HA and the globular head domain of the cH5/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/turkey/Turkey/1/2005 (H5) HA. In another specific embodiment, the stemdomain of a cH5/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Perth/16/2009 (H3N2) HA and the globular head domain ofthe cH5/3 chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/Whooperswan/Mongolia/244/2005 (H5) HA.

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza virus of the H3 subtype and (ii) theglobular head domain of the hemagglutinin from an influenza virus of theH7 subtype (sometimes referred to herein as a “cH7/3 chimeric influenzahemagglutinin polypeptide”). In a specific embodiment, the stem domainof a cH7/3 chimeric influenza hemagglutinin polypeptide is the stemdomain of A/Victoria/361/2011 (H3N2) HA. In another specific embodiment,the stem domain of a cH7/3 chimeric influenza hemagglutinin polypeptideis the stem domain of A/Victoria/361/2011 (H3N2) HA and the globularhead domain of the cH7/3 chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Netherlands/219/03 (H7) HA. In anotherspecific embodiment, the stem domain of a cH7/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/Victoria/361/2011(H3N2) HA and the globular head domain of the cH7/3 chimeric influenzahemagglutinin polypeptide is the globular head domain of A/Canada/504/04(H7) HA. In another specific embodiment, the stem domain of a cH7/3chimeric influenza hemagglutinin polypeptide is the stem domain ofA/Victoria/361/2011 (H3N2) HA and the globular head domain of the cH7/3chimeric influenza hemagglutinin polypeptide is the globular head domainof A/Canada/444/04 (H7) HA. In another specific embodiment, the stemdomain of a cH7/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Victoria/361/2011 (H3N2) HA and the globular headdomain of the cH7/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/chicken/Jalisco/CPA1/2012 (H7) HA. In anotherspecific embodiment, the stem domain of a cH7/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/Victoria/361/2011(H3N2) HA and the globular head domain of the cH7/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/mallard/Alberta/24/2001 (H7) HA. In another specific embodiment, thestem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide isthe stem domain of A/Victoria/361/2011 (H3N2) HA and the globular headdomain of the cH7/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Rhea/NC/39482/93 (H7) HA. In another specificembodiment, the stem domain of a cH7/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Victoria/361/2011 (H3N2) HA and theglobular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/mallard/Netherlands/12/2000(H7) HA.

In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA. In another specific embodiment, thestem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide isthe stem domain of A/harbor seal/Massachusetts/1/2011 (H3N8) HA and theglobular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Netherlands/219/03 (H7) HA.In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Canada/504/04 (H7) HA. In another specific embodiment, thestem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide isthe stem domain of A/harbor seal/Massachusetts/1/2011 (H3N8) HA and theglobular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Canada/444/04 (H7) HA. Inanother specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/chicken/Jalisco/CPA1/2012 (H7) HA. In another specificembodiment, the stem domain of a cH7/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/harbor seal/Massachusetts/1/2011(H3N8) HA and the globular head domain of the cH7/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/mallard/Alberta/24/2001 (H7) HA. In another specific embodiment, thestem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide isthe stem domain of A/harbor seal/Massachusetts/1/2011 (H3N8) HA and theglobular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Rhea/NC/39482/93 (H7) HA.In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain of A/harborseal/Massachusetts/1/2011 (H3N8) HA and the globular head domain of thecH7/3 chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/mallard/Netherlands/12/2000 (H7) HA.

In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA. In another specific embodiment, the stemdomain of a cH7/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Indiana/10/2011 (H3N2) HA and the globular head domainof the cH7/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Netherlands/219/03 (H7) HA. In anotherspecific embodiment, the stem domain of a cH7/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/Indiana/10/2011 (H3N2)HA and the globular head domain of the cH7/3 chimeric influenzahemagglutinin polypeptide is the globular head domain of A/Canada/504/04(H7) HA. In another specific embodiment, the stem domain of a cH7/3chimeric influenza hemagglutinin polypeptide is the stem domain ofA/Indiana/10/2011 (H3N2) HA and the globular head domain of the cH7/3chimeric influenza hemagglutinin polypeptide is the globular head domainof A/Canada/444/04 (H7) HA. In another specific embodiment, the stemdomain of a cH7/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Indiana/10/2011 (H3N2) HA and the globular head domainof the cH7/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/chicken/Jalisco/CPA1/2012 (H7) HA. In anotherspecific embodiment, the stem domain of a cH7/3 chimeric influenzahemagglutinin polypeptide is the stem domain of A/Indiana/10/2011 (H3N2)HA and the globular head domain of the cH7/3 chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/mallard/Alberta/24/2001 (H7) HA. In another specific embodiment, thestem domain of a cH7/3 chimeric influenza hemagglutinin polypeptide isthe stem domain of A/Indiana/10/2011 (H3N2) HA and the globular headdomain of the cH7/3 chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Rhea/NC/39482/93 (H7) HA. In another specificembodiment, the stem domain of a cH7/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Indiana/10/2011 (H3N2) HA and theglobular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/mallard/Netherlands/12/2000(H7) HA.

In another specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA. In another specific embodiment, the stemdomain of a cH7/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Perth/16/2009 (H3N2) HA and the globular head domain ofthe cH7/3 chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/Netherlands/219/03 (H7) HA. In another specificembodiment, the stem domain of a cH7/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Perth/16/2009 (H3N2) HA and theglobular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Canada/504/04 (H7) HA. Inanother specific embodiment, the stem domain of a cH7/3 chimericinfluenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA and the globular head domain of the cH7/3chimeric influenza hemagglutinin polypeptide is the globular head domainof A/Canada/444/04 (H7) HA. In another specific embodiment, the stemdomain of a cH7/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Perth/16/2009 (H3N2) HA and the globular head domain ofthe cH7/3 chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/chicken/Jalisco/CPA1/2012 (H7) HA. In another specificembodiment, the stem domain of a cH7/3 chimeric influenza hemagglutininpolypeptide is the stem domain of A/Perth/16/2009 (H3N2) HA and theglobular head domain of the cH7/3 chimeric influenza hemagglutininpolypeptide is the globular head domain of A/mallard/Alberta/24/2001(H7) HA. In another specific embodiment, the stem domain of a cH7/3chimeric influenza hemagglutinin polypeptide is the stem domain ofA/Perth/16/2009 (H3N2) HA and the globular head domain of the cH7/3chimeric influenza hemagglutinin polypeptide is the globular head domainof A/Rhea/NC/39482/93 (H7) HA. In another specific embodiment, the stemdomain of a cH7/3 chimeric influenza hemagglutinin polypeptide is thestem domain of A/Perth/16/2009 (H3N2) HA and the globular head domain ofthe cH7/3 chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/mallard/Netherlands/12/2000 (H7) HA.

In a specific embodiment, a cH7/3 chimeric influenza hemagglutininpolypeptide provided herein does not comprise the globular head domainof A/mallard/Alberta/24/2001 (H7) HA. In another specific embodiment, acH7/3 chimeric influenza hemagglutinin polypeptide does not comprise thestem domain of A/Perth/16/2009 (H3) HA.

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza B virus and (ii) the globular headdomain of the hemagglutinin from an influenza virus of the H5 subtype(sometimes referred to herein as a “cH5/B chimeric influenzahemagglutinin polypeptide”). In a specific embodiment, the stem domainof a cH5/B chimeric influenza hemagglutinin polypeptide is the stemdomain of B/Malaysia/2506/2004 HA. In another specific embodiment, thestem domain of a cH5/B chimeric influenza hemagglutinin polypeptide isthe stem domain of B/Malaysia/2506/2004 HA and the globular head domainof the cH5/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Vietnam/1203/2004 (H5) HA. In another specificembodiment, the stem domain of a cH5/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Malaysia/2506/2004 HA and theglobular head domain of the cH5/B chimeric influenza hemagglutininpolypeptide is the globular head domain of A/Indonesia/5/2005 (H5) HA.In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Malaysia/2506/2004 HA and the globular head domain of the cH5/Bchimeric influenza hemagglutinin polypeptide is the globular head domainof A/Anhui/1/2005 (H5) HA. In another specific embodiment, the stemdomain of a cH5/B chimeric influenza hemagglutinin polypeptide is thestem domain of B/Malaysia/2506/2004 HA and the globular head domain ofthe cH5/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA. In anotherspecific embodiment, the stem domain of a cH5/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HAand the globular head domain of the cH5/B chimeric influenzahemagglutinin polypeptide is the globular head domain ofA/turkey/Turkey/1/2005 (H5) HA. In another specific embodiment, the stemdomain of a cH5/B chimeric influenza hemagglutinin polypeptide is thestem domain of B/Malaysia/2506/2004 HA and the globular head domain ofthe cH5/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/Whooperswan/Mongolia/244/2005 (H5) HA.

In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA. In another specific embodiment, the stem domain ofa cH5/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Florida/4/2006 HA and the globular head domain of the cH5/Bchimeric influenza hemagglutinin polypeptide is the globular head domainof A/Vietnam/1203/2004 (H5) HA. In another specific embodiment, the stemdomain of a cH5/B chimeric influenza hemagglutinin polypeptide is thestem domain of B/Florida/4/2006 HA and the globular head domain of thecH5/B chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Indonesia/5/2005 (H5) HA. In another specific embodiment,the stem domain of a cH5/B chimeric influenza hemagglutinin polypeptideis the stem domain of B/Florida/4/2006 HA and the globular head domainof the cH5/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Anhui/1/2005 (H5) HA. In another specificembodiment, the stem domain of a cH5/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Florida/4/2006 HA and the globularhead domain of the cH5/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA.In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/turkey/Turkey/1/2005 (H5) HA. In another specific embodiment, the stemdomain of a cH5/B chimeric influenza hemagglutinin polypeptide is thestem domain of B/Florida/4/2006 and the globular head domain of thecH5/B chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Whooperswan/Mongolia/244/2005 (H5) HA.

In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA. In another specific embodiment, the stem domainof a cH5/B chimeric influenza hemagglutinin polypeptide is the stemdomain of B/Wisconsin/1/2010 HA and the globular head domain of thecH5/B chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Vietnam/1203/2004 (H5) HA. In another specific embodiment,the stem domain of a cH5/B chimeric influenza hemagglutinin polypeptideis the stem domain of B/Wisconsin/1/2010 HA and the globular head domainof the cH5/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Indonesia/5/2005 (H5) HA. In another specificembodiment, the stem domain of a cH5/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Wisconsin/1/2010 HA and the globularhead domain of the cH5/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Anhui/1/2005 (H5) HA. In another specificembodiment, the stem domain of a cH5/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Wisconsin/1/2010 HA and the globularhead domain of the cH5/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA.In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/turkey/Turkey/1/2005 (H5) HA. In another specific embodiment, the stemdomain of a cH5/B chimeric influenza hemagglutinin polypeptide is thestem domain of B/Wisconsin/1/2010 HA and the globular head domain of thecH5/B chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Whooperswan/Mongolia/244/2005 (H5) HA.

In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA. In another specific embodiment, the stem domainof a cH5/B chimeric influenza hemagglutinin polypeptide is the stemdomain of B/Brisbane/60/2008 HA and the globular head domain of thecH5/B chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Vietnam/1203/2004 (H5) HA. In another specific embodiment,the stem domain of a cH5/B chimeric influenza hemagglutinin polypeptideis the stem domain of B/Brisbane/60/2008 HA and the globular head domainof the cH5/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Indonesia/5/2005 (H5) HA. In another specificembodiment, the stem domain of a cH5/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Brisbane/60/2008 HA and the globularhead domain of the cH5/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Anhui/1/2005 (H5) HA. In another specificembodiment, the stem domain of a cH5/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Brisbane/60/2008 HA and the globularhead domain of the cH5/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Bar headed goose/Quinghai/1A/2005 (H5) HA.In another specific embodiment, the stem domain of a cH5/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA and the globular head domain of the cH5/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/turkey/Turkey/1/2005 (H5) HA. In another specific embodiment, the stemdomain of a cH5/B chimeric influenza hemagglutinin polypeptide is thestem domain of B/Brisbane/60/2008 HA and the globular head domain of thecH5/B chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Whooperswan/Mongolia/244/2005 (H5) HA.

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza B virus and (ii) the globular headdomain of the hemagglutinin from an influenza virus of the H7 subtype(sometimes referred to herein as a “cH7/B chimeric influenzahemagglutinin polypeptide”). In a specific embodiment, the stem domainof a cH7/B chimeric influenza hemagglutinin polypeptide is the stemdomain of B/Malaysia/2506/2004 HA. In another specific embodiment, thestem domain of a cH7/B chimeric influenza hemagglutinin polypeptide isthe stem domain of B/Malaysia/2506/2004 HA and the globular head domainof the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Netherlands/219/03 (H7) HA. In anotherspecific embodiment, the stem domain of a cH7/B chimeric influenzahemagglutinin polypeptide is the stem domain of B/Malaysia/2506/2004 HAand the globular head domain of the cH7/B chimeric influenzahemagglutinin polypeptide is the globular head domain of A/Canada/504/04(H7) HA. In another specific embodiment, the stem domain of a cH7/Bchimeric influenza hemagglutinin polypeptide is the stem domain ofB/Malaysia/2506/2004 HA and the globular head domain of the cH7/Bchimeric influenza hemagglutinin polypeptide is the globular head domainof A/Canada/444/04 (H7) HA. In another specific embodiment, the stemdomain of a cH7/B chimeric influenza hemagglutinin polypeptide is thestem domain of B/Malaysia/2506/2004 HA and the globular head domain ofthe cH7/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/chicken/Jalisco/CPA1/2012 (H7) HA. In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Malaysia/2506/2004 HA and theglobular head domain of the cH7/B chimeric influenza hemagglutininpolypeptide is the globular head domain of A/mallard/Alberta/24/2001(H7) HA. In another specific embodiment, the stem domain of a cH7/Bchimeric influenza hemagglutinin polypeptide is the stem domain ofB/Malaysia/2506/2004 HA and the globular head domain of the cH7/Bchimeric influenza hemagglutinin polypeptide is the globular head domainof A/Rhea/NC/39482/93 (H7) HA. In another specific embodiment, the stemdomain of a cH7/B chimeric influenza hemagglutinin polypeptide is thestem domain of B/Malaysia/2506/2004 HA and the globular head domain ofthe cH7/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/mallard/Massachusetts/12/2000 (H7) HA.

In another specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA. In another specific embodiment, the stem domain ofa cH7/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Florida/4/2006 HA and the globular head domain of the cH7/Bchimeric influenza hemagglutinin polypeptide is the globular head domainof A/Netherlands/219/03 (H7) HA. In another specific embodiment, thestem domain of a cH7/B chimeric influenza hemagglutinin polypeptide isthe stem domain of B/Florida/4/2006 HA and the globular head domain ofthe cH7/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/Canada/504/04 (H7) HA. In another specific embodiment,the stem domain of a cH7/B chimeric influenza hemagglutinin polypeptideis the stem domain of B/Florida/4/2006 HA and the globular head domainof the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Canada/444/04 (H7) HA. In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Florida/4/2006 HA and the globularhead domain of the cH7/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/chicken/Jalisco/CPA1/2012 (H7) HA. Inanother specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA and the globular head domain of the cH7/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/mallard/Alberta/24/2001 (H7) HA. In another specific embodiment, thestem domain of a cH7/B chimeric influenza hemagglutinin polypeptide isthe stem domain of B/Florida/4/2006 HA and the globular head domain ofthe cH7/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/Rhea/NC/39482/93 (H7) HA. In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Florida/4/2006 HA and the globularhead domain of the cH7/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/mallard/Massachusetts/12/2000 (H7) HA.

In another specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA. In another specific embodiment, the stem domainof a cH7/B chimeric influenza hemagglutinin polypeptide is the stemdomain of B/Wisconsin/1/2010 HA and the globular head domain of thecH7/B chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Netherlands/219/03 (H7) HA. In another specific embodiment,the stem domain of a cH7/B chimeric influenza hemagglutinin polypeptideis the stem domain of B/Wisconsin/1/2010 HA and the globular head domainof the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Canada/504/04 (H7) HA. In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Wisconsin/1/2010 HA and the globularhead domain of the cH7/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Canada/444/04 (H7) HA. In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Wisconsin/1/2010 HA and the globularhead domain of the cH7/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/chicken/Jalisco/CPA1/2012 (H7) HA. Inanother specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA and the globular head domain of the cH7/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/mallard/Alberta/24/2001 (H7) HA. In another specific embodiment, thestem domain of a cH7/B chimeric influenza hemagglutinin polypeptide isthe stem domain of B/Wisconsin/1/2010 HA and the globular head domain ofthe cH7/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/Rhea/NC/39482/93 (H7) HA. In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Wisconsin/1/2010 HA and the globularhead domain of the cH7/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/mallard/Massachusetts/12/2000 (H7) HA.

In another specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA. In another specific embodiment, the stem domainof a cH7/B chimeric influenza hemagglutinin polypeptide is the stemdomain of B/Brisbane/60/2008 HA and the globular head domain of thecH7/B chimeric influenza hemagglutinin polypeptide is the globular headdomain of A/Netherlands/219/03 (H7) HA. In another specific embodiment,the stem domain of a cH7/B chimeric influenza hemagglutinin polypeptideis the stem domain of B/Brisbane/60/2008 HA and the globular head domainof the cH7/B chimeric influenza hemagglutinin polypeptide is theglobular head domain of A/Canada/504/04 (H7) HA. In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Brisbane/60/2008 HA and the globularhead domain of the cH7/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/Canada/444/04 (H7) HA. In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Brisbane/60/2008 HA and the globularhead domain of the cH7/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/chicken/Jalisco/CPA1/2012 (H7) HA. Inanother specific embodiment, the stem domain of a cH7/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA and the globular head domain of the cH7/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofA/mallard/Alberta/24/2001 (H7) HA. In another specific embodiment, thestem domain of a cH7/B chimeric influenza hemagglutinin polypeptide isthe stem domain of B/Brisbane/60/2008 HA and the globular head domain ofthe cH7/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of A/Rhea/NC/39482/93 (H7) HA. In another specificembodiment, the stem domain of a cH7/B chimeric influenza hemagglutininpolypeptide is the stem domain of B/Brisbane/60/2008 HA and the globularhead domain of the cH7/B chimeric influenza hemagglutinin polypeptide isthe globular head domain of A/mallard/Massachusetts/12/2000 (H7) HA.

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide provided herein comprises (i) the stem domain of thehemagglutinin from an influenza B virus and (ii) the globular headdomain of the hemagglutinin from a different influenza B virus strain(sometimes referred to herein as a “cB/B chimeric influenzahemagglutinin polypeptide”). In a specific embodiment, the stem domainof a cB/B chimeric influenza hemagglutinin polypeptide is the stemdomain of B/Malaysia/2506/2004 HA. In another specific embodiment, thestem domain of a cB/B chimeric influenza hemagglutinin polypeptide isthe stem domain of B/Malaysia/2506/2004 HA and the globular head domainof the cB/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of B/Lee/1940 HA. In another specific embodiment, the stemdomain of a cB/B chimeric influenza hemagglutinin polypeptide is thestem domain of B/Malaysia/2506/2004 HA and the globular head domain ofthe cB/B chimeric influenza hemagglutinin polypeptide is the globularhead domain of B/seal/Netherlands/1/99 HA (or aB/seal/Netherlands/1/99-like influenza virus).

In another specific embodiment, the stem domain of a cB/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA. In another specific embodiment, the stem domain ofa cB/B chimeric influenza hemagglutinin polypeptide is the stem domainof B/Florida/4/2006 HA and the globular head domain of the cB/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofB/Lee/1940 HA. In another specific embodiment, the stem domain of a cB/Bchimeric influenza hemagglutinin polypeptide is the stem domain ofB/Florida/4/2006 HA and the globular head domain of the cB/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofB/seal/Netherlands/1/99 HA (or a B/seal/Netherlands/1/99-like influenzavirus).

In another specific embodiment, the stem domain of a cB/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA. In another specific embodiment, the stem domainof a cB/B chimeric influenza hemagglutinin polypeptide is the stemdomain of B/Wisconsin/1/2010 HA and the globular head domain of the cB/Bchimeric influenza hemagglutinin polypeptide is the globular head domainof B/Lee/1940 HA. In another specific embodiment, the stem domain of acB/B chimeric influenza hemagglutinin polypeptide is the stem domain ofB/Wisconsin/1/2010 HA and the globular head domain of the cB/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofB/seal/Netherlands/1/99 HA (or a B/seal/Netherlands/1/99-like influenzavirus).

In another specific embodiment, the stem domain of a cB/B chimericinfluenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA. In another specific embodiment, the stem domainof a cB/B chimeric influenza hemagglutinin polypeptide is the stemdomain of B/Brisbane/60/2008 HA and the globular head domain of the cB/Bchimeric influenza hemagglutinin polypeptide is the globular head domainof B/Lee/1940 HA. In another specific embodiment, the stem domain of acB/B chimeric influenza hemagglutinin polypeptide is the stem domain ofB/Brisbane/60/2008 HA and the globular head domain of the cB/B chimericinfluenza hemagglutinin polypeptide is the globular head domain ofB/seal/Netherlands/1/99 HA (or a B/seal/Netherlands/1/99-like influenzavirus).

In certain embodiments, a chimeric influenza virus hemagglutininpolypeptide provided herein is monomeric. In certain embodiments, achimeric influenza virus hemagglutinin polypeptide provided herein ismultimeric. In certain embodiments, a chimeric influenza virushemagglutinin polypeptide provided herein is trimeric.

In certain embodiments, a chimeric influenza virus hemagglutininpolypeptide provided herein comprises a signal peptide. Typically, thesignal peptide is cleaved during or after polypeptide expression andtranslation to yield a mature chimeric influenza virus hemagglutininpolypeptide. In certain embodiments, also provided herein are maturechimeric influenza virus hemagglutinin polypeptides that lack a signalpeptide. In embodiments where a chimeric influenza virus hemagglutininpolypeptide provided herein comprises a signal peptide, the signalpeptide might be based on any influenza virus signal peptide known tothose of skill in the art. In certain embodiments, the signal peptidesare based on influenza A signal peptides. In certain embodiments, thesignal peptides are based on the signal peptide of an influenza Ahemagglutinin selected from the group consisting of H1, H2, H3, H4, H5,H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, and H17. In certainembodiments, the signal peptide might be any signal peptide deemeduseful to one of skill in the art.

In certain embodiments, a chimeric influenza virus hemagglutininpolypeptide provided herein comprises a luminal domain. In embodimentswhere a chimeric influenza virus hemagglutinin polypeptide providedherein comprises a luminal domain, the luminal domain might be based onany influenza luminal domain known to those of skill in the art. Incertain embodiments, the luminal domains are based on influenza Aluminal domains. In certain embodiments, the luminal domains are basedon the luminal domain of an influenza A hemagglutinin selected from thegroup consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12,H13, H14, H15, H16, and H17. In certain embodiments, the luminal domainmight be any luminal domain deemed useful to one of skill in the art. Incertain embodiments, the luminal domains are from the same hemagglutininas the stem domain. In certain embodiments, the luminal domains are frominfluenza virus strain or subtype as the stem domain HA2 subunit.

In certain embodiments, a chimeric influenza virus hemagglutininpolypeptide provided herein comprises a transmembrane domain. Inembodiments where a chimeric influenza virus hemagglutinin polypeptideprovided herein comprises a transmembrane domain, the transmembranedomain might be based on any influenza transmembrane domain known tothose of skill in the art. In certain embodiments, the transmembranedomains are based on influenza A transmembrane domains. In certainembodiments, the transmembrane domains are based on a transmembranedomain of an influenza A hemagglutinin selected from the groupconsisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13,H14, H15, H16, and H17. In certain embodiments, the transmembrane domainmight be any transmembrane domain deemed useful to one of skill in theart. In certain embodiments, the transmembrane domains are from the samehemagglutinin as the stem domain. In certain embodiments, thetransmembrane domains are from influenza virus strain or subtype as thestem domain HA2 subunit.

In certain embodiments, a chimeric influenza virus hemagglutininpolypeptide provided herein comprises a cytoplasmic domain. Inembodiments where a chimeric influenza virus hemagglutinin polypeptideprovided herein comprises a cytoplasmic domain, the cytoplasmic domainmight be based on any influenza cytoplasmic domain known to those ofskill in the art. In certain embodiments, the cytoplasmic domains arebased on influenza A cytoplasmic domains. In certain embodiments, thecytoplasmic domains are based on a cytoplasmic domain of an influenza Ahemagglutinin selected from the group consisting of H1, H2, H3, H4, H5,H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, and H17. In certainembodiments, the cytoplasmic domain might be any cytoplasmic domaindeemed useful to one of skill in the art. In certain embodiments, thecytoplasmic domains are from the same hemagglutinin as the stem domain.In certain embodiments, the cytoplasmic domains are from influenza virusstrain or subtype as the stem domain HA2 subunit.

In certain embodiments, the chimeric influenza virus hemagglutininpolypeptides provided herein further comprise one or more polypeptidedomains. Useful polypeptide domains include domains that facilitatepurification, folding and cleavage of portions of a polypeptide. Forexample, a His tag (His-His-His-His-His-His, SEQ ID NO:17), FLAG epitopeor other purification tag can facilitate purification of a chimericinfluenza virus hemagglutinin polypeptide provided herein. In someembodiments, the His tag has the sequence, (His)_(n), wherein n is 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or greater.A foldon, or trimerization, domain from bacteriophage T4 fibritin canfacilitate trimerization of polypeptides provided herein. In someembodiments, the trimerization domain comprises a wildtype GCN4pIItrimerization heptad repeat or a modified GCN4pII trimerization heptadrepeat that allows for the formation of trimeric or tetrameric coiledcoils. See, e.g., Weldon et al., 2010, PLoSONE 5(9): e12466. The foldondomain can have any foldon sequence known to those of skill in the art(see, e.g., Papanikolopoulou et al., 2004, J. Biol. Chem.279(10):8991-8998, the contents of which are hereby incorporated byreference in their entirety). Examples includeGSGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:18). A foldon domain can beuseful to facilitate trimerization of soluble polypeptides providedherein. Cleavage sites can be used to facilitate cleavage of a portionof a polypeptide, for example cleavage of a purification tag or foldondomain or both. Useful cleavage sites include a thrombin cleavage site,for example one with the sequence LVPRGSP (SEQ ID NO:19). In certainembodiments, the cleavage site is a cleavage site recognized by TobaccoEtch Virus (TEV) protease (e.g., amino acid sequenceGlu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) (SEQ ID NO:20)).

In certain embodiments, the chimeric influenza hemagglutininpolypeptides described herein are soluble polypeptides.

In certain embodiments, the influenza hemagglutinin stem domainpolypeptides of the chimeric influenza virus hemagglutinin polypeptidesdescribed herein maintain the cysteine residues identified in influenzahemagglutinin polypeptides as A_(p) and A_(q) in FIG. 1, i.e., thecysteine residues identified in influenza hemagglutinin polypeptides asA_(p) and A_(q) in FIG. 1 are maintained in the chimeric influenza virushemagglutinin polypeptides described herein. Thus, in certainembodiments, in the primary sequence of a chimeric influenza virushemagglutinin polypeptide described herein: (i) the N-terminal segmentof an influenza hemagglutinin stem domain polypeptide ends at thecysteine residue identified as A_(p) in FIG. 1, (ii) the C-terminalsegment of an influenza hemagglutinin stem domain polypeptide begins atthe cysteine residue identified as A_(q) in FIG. 1; and (iii) theinfluenza hemagglutinin head domain polypeptide (which is heterologousto the influenza hemagglutinin stem domain polypeptide) is between theN-terminal and C-terminal segments of the influenza hemagglutinin stemdomain polypeptide.

In certain embodiments, the HA1 N-terminal stem segment of the chimericinfluenza virus hemagglutinin polypeptides described herein does not endexactly at A_(p) (e.g., Cys₅₂ of an HA1 subunit from an H3hemagglutinin), but at a residue in sequence and structural vicinity toA_(p). For example, in certain embodiments, the HA1 N-terminal stemsegment of the chimeric influenza virus hemagglutinin polypeptidesdescribed herein ends at A_(p−1), A_(p−2), A_(p−3), A_(p−4), A_(p−5),A_(p−6), A_(p−7), A_(p−8), A_(p−9), A_(p−10). In certain embodiments,the HA1 N-terminal stem segment of the chimeric influenza virushemagglutinin polypeptides described herein ends in the range of A_(p−1)to A_(p−3), A_(p−3) to A_(p−5), A_(p−5) to A_(p−8), A_(p−8) to A_(p−10).For example, an HA1 N-terminal stem segment ending at A_(p−10) would endat Lys42 of an H3 hemagglutinin. In certain embodiments, the HA1N-terminal stem segment of the chimeric influenza virus hemagglutininpolypeptides described herein ends at A_(p+1), A_(p+2), A_(p+3),A_(p+4), A_(p+5), A_(p+6), A_(p+7), A_(p+8), A_(p+9), A_(p+10). Incertain embodiments, the HA1 N-terminal stem segment of the chimericinfluenza virus hemagglutinin polypeptides described herein ends in therange of A_(p+1) to A_(p+5), A_(p+5) to A_(p+10). The end of an HA1N-terminal stem segment should be selected in conjunction with the endof the HA1 C-terminal stem segment and the influenza hemagglutinin headdomain polypeptide so that the resulting chimeric influenza virushemagglutinin polypeptide is capable of forming a three-dimensionalstructure similar to a wild-type influenza hemagglutinin. In suchembodiments, an influenza hemagglutinin head domain polypeptide (whichis heterologous to the influenza hemagglutinin stem domain polypeptide)is located, in primary sequence, between the N-terminal and C-terminalsegments of the influenza hemagglutinin stem domain polypeptide.

In certain embodiments, the HA1 C-terminal stem segment of the chimericinfluenza virus hemagglutinin polypeptides described herein does notstart exactly at A_(q) (e.g., Cys₂₇₇ of an HA1 subunit from an H3hemagglutinin), but at a residue in sequence and structural vicinity toA_(q). For example, in certain embodiments, the HA1 C-terminal stemsegment of the chimeric influenza virus hemagglutinin polypeptidesdescribed herein starts at about A_(q−1), A_(q−2), A_(q−3), A_(q−4),A_(q−5), A_(q−6), A_(q−7), A_(q−8), A_(q−9), A_(q−10). In certainembodiments, the HA1 C-terminal stem segment of the chimeric influenzavirus hemagglutinin polypeptides described herein starts in the range ofA_(q−1) to A_(q−5), A_(q−5) to A_(q−10). For example, an HA1 C-terminalstem segment ending at A_(q−10) would start at Isoleucine262 of an H3hemagglutinin. In certain embodiments, the HA1 C-terminal stem segmentof the chimeric influenza virus hemagglutinin polypeptides describedherein starts at A_(q+1), A_(q+2), A_(q+3), A_(q+4), A_(q+5), A_(q+6),A_(q+7), A_(q+8), A_(q+9), A_(q+10). In certain embodiments, the HA1C-terminal stem segment of the chimeric influenza virus hemagglutininpolypeptides described herein starts in the range of A_(q+1) to A_(q+3),A_(q+3) to A_(q+5), A_(q+5) to A_(q+8), A_(q+8) to A_(q+10). The end ofan HA1 N-terminal stem segment should be selected in conjunction withthe start of the HA1 C-terminal stem segment and the influenzahemagglutinin head domain polypeptide so that the resulting chimericinfluenza virus hemagglutinin polypeptide is capable of forming athree-dimensional structure similar to a wild-type influenzahemagglutinin. In such embodiments, an influenza hemagglutinin headdomain polypeptide (which is heterologous to the influenza hemagglutininstem domain polypeptide) is located, in primary sequence, between theN-terminal and C-terminal segments of the influenza hemagglutinin stemdomain polypeptide.

In one example, an HA1 N-terminal stem segment of a chimeric influenzavirus hemagglutinin polypeptide described herein may end at any one ofhemagglutinin amino acid positions 45-48 (using H3 numbering) and an HA1C-terminal stem segment of the chimeric influenza virus hemagglutininpolypeptide may start at any one of hemagglutinin amino acid positions282-287 (using H3 numbering); and the heterologous head domain may beginat any one of amino acid positions 46-49 and end at any one of aminoacid position 284-289 (using H3 numbering).

In certain embodiments, when the stem domain of the chimeric influenzavirus hemagglutinin polypeptides described herein is derived from aninfluenza B virus, the HA1 N-terminal stem segment may end at, e.g.,amino acid 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 of the HA (in primary amino acid sequence), and the HA1 C-terminalstem segment may begin at, e.g., amino acid 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, or 300 of the HA (in primary amino acid sequence). The globularhead domain of the chimeric influenza virus hemagglutinin polypeptidethus would be inserted between the HA1 N-terminal stem segment and theHA1 C-terminal stem segment of the influenza B virus stem domain. Forexample, in the HA of influenza virus B/Hong Kong/8/73(PDB:2RFT:GenBank:M10298.1), the HA1 N-terminal stem segment would begin withamino acids DRICT (SEQ ID NO:22), with “D” being position 1, and wouldend at amino acid position 42 (in primary sequence); and the HA1C-terminal stem segment would begin at amino acid position 288 (inprimary sequence) and continue to the end of the C-terminus of the HA.

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide described herein may be conjugated to heterologous proteins,e.g., a major histocompatibility complex (MHC) with or without heatshock proteins (e.g., Hsp10, Hsp20, Hsp30, Hsp40, Hsp60, Hsp70, Hsp90,or Hsp100). In certain embodiments, a chimeric influenza hemagglutinin(HA) polypeptide described herein may be conjugated to immunomodulatorymolecules, such as proteins which would target the chimeric influenzahemagglutinin (HA) polypeptide to immune cells such as B cells (e.g.,C3d) or T cells. In certain embodiments, chimeric influenzahemagglutinin (HA) polypeptide described herein may be conjugated toproteins which stimulate the innate immune system such as interferontype 1, alpha, beta, or gamma interferon, colony stimulating factorssuch as granulocyte-macrophage colony-stimulating factor (GM-CSF),interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18,IL-21, IL-23, tumor necrosis factor (TNF)-β, TNFα, B7.1, B7.2, 4-1BB,CD40 ligand (CD40L), and drug-inducible CD40 (iCD40).

It will be understood by those of skill in the art that the chimericinfluenza virus hemagglutinin polypeptides provided herein can beprepared according to any technique known by and deemed suitable tothose of skill in the art, including the techniques described herein. Incertain embodiments, the chimeric influenza virus hemagglutininpolypeptides are isolated.

5.2 Nucleic Acids Encoding Chimeric Influenza Virus Hemagglutinin (HA)Polypeptides

Provided herein are nucleic acids that encode the chimeric influenzahemagglutinin (HA) polypeptides described herein (e.g., the chimericinfluenza hemagglutinin (HA) polypeptides described in Section 5.1). Dueto the degeneracy of the genetic code, any nucleic acid that encodes achimeric influenza hemagglutinin (HA) polypeptide described herein isencompassed herein. In certain embodiments, nucleic acids correspondingto, or capable of hybridizing with, naturally occurring influenza virusnucleic acids encoding an HA1 N-terminal stem segment, an HA1 C-terminalstem segment, HA2 domain, luminal domain, transmembrane domain, and/orcytoplasmic domain are used to produce a chimeric influenzahemagglutinin (HA) polypeptide described herein.

Also provided herein are nucleic acids capable of hybridizing to anucleic acid encoding a chimeric influenza hemagglutinin (HA)polypeptide described herein (e.g., the chimeric influenza hemagglutinin(HA) polypeptides described in Section 5.1). In certain embodiments,provided herein are nucleic acids capable of hybridizing to a fragmentof a nucleic acid encoding a chimeric influenza hemagglutinin (HA)polypeptide described herein. In other embodiments, provided herein arenucleic acids capable of hybridizing to the full length nucleic acidencoding a chimeric influenza hemagglutinin (HA) polypeptide describedherein. General parameters for hybridization conditions for nucleicacids are described in Sambrook et al., Molecular Cloning—A LaboratoryManual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989), and in Ausubel et al., Current Protocols inMolecular Biology, vol. 2, Current Protocols Publishing, New York(1994). Hybridization may be performed under high stringency conditions,medium stringency conditions, or low stringency conditions. Those ofskill in the art will understand that low, medium and high stringencyconditions are contingent upon multiple factors all of which interactand are also dependent upon the nucleic acids in question. For example,high stringency conditions may include temperatures within 5° C. meltingtemperature of the nucleic acid(s), a low salt concentration (e.g., lessthan 250 mM), and a high co-solvent concentration (e.g., 1-20% ofco-solvent, e.g., DMSO). Low stringency conditions, on the other hand,may include temperatures greater than 10° C. below the meltingtemperature of the nucleic acid(s), a high salt concentration (e.g.,greater than 1000 mM) and the absence of co-solvents.

In some embodiments, a nucleic acid encoding a chimeric influenzahemagglutinin (HA) polypeptide described herein is isolated. In certainembodiments, an “isolated” nucleic acid refers to a nucleic acidmolecule which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. In other words, theisolated nucleic acid can comprise heterologous nucleic acids that arenot associated with it in nature. In other embodiments, an “isolated”nucleic acid, such as a cDNA molecule, can be substantially free ofother cellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. The term “substantially free ofcellular material” includes preparations of nucleic acid in which thenucleic acid is separated from cellular components of the cells fromwhich it is isolated or recombinantly produced. Thus, nucleic acid thatis substantially free of cellular material includes preparations ofnucleic acid having less than about 30%, 20%, 10%, or 5% (by dry weight)of other nucleic acids. The term “substantially free of culture medium”includes preparations of nucleic acid in which the culture mediumrepresents less than about 50%, 20%, 10%, or 5% of the volume of thepreparation. The term “substantially free of chemical precursors orother chemicals” includes preparations in which the nucleic acid isseparated from chemical precursors or other chemicals which are involvedin the synthesis of the nucleic acid. In specific embodiments, suchpreparations of the nucleic acid have less than about 50%, 30%, 20%,10%, 5% (by dry weight) of chemical precursors or compounds other thanthe nucleic acid of interest.

In addition, provided herein are nucleic acids encoding the individualcomponents of a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein, e.g., nucleic acids encoding the globular head domain,the HA1 N-terminal stem segment, the HA1 C-terminal stem segment and/orthe HA2 domain a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein are provided herein. Nucleic acids encoding componentsof a chimeric influenza hemagglutinin (HA) polypeptide described hereinmay be assembled using standard molecular biology techniques known tothe one of skill in the art so as to yield a chimeric influenzahemagglutinin (HA) polypeptide described herein.

5.3 Expression of Chimeric Influenza Virus Hemagglutinin (HA)Polypeptides

Provided herein are vectors, including expression vectors, containing anucleic acid encoding a chimeric influenza hemagglutinin (HA)polypeptide described herein (e.g., the chimeric influenza hemagglutinin(HA) polypeptides described in Section 5.1). In a specific embodiment,the vector is an expression vector that is capable of directing theexpression of a nucleic acid encoding a chimeric influenza hemagglutinin(HA) polypeptide described herein. Non-limiting examples of expressionvectors include, but are not limited to, plasmids and viral vectors,such as replication defective retroviruses, adenoviruses,adeno-associated viruses and baculoviruses. Expression vectors also mayinclude, without limitation, transgenic animals and non-mammaliancells/organisms, e.g., mammalian cells/organisms that have beenengineered to perform mammalian N-linked glycosylation.

In some embodiments, provided herein are expression vectors encodingcomponents of a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein. Such vectors may be used to express the components inone or more host cells and the components may be isolated and conjugatedtogether with a linker using techniques known to one of skill in theart.

An expression vector comprises a nucleic acid encoding a chimericinfluenza hemagglutinin (HA) polypeptide described herein in a formsuitable for expression of the nucleic acid in a host cell. In aspecific embodiment, an expression vector includes one or moreregulatory sequences, selected on the basis of the host cells to be usedfor expression, which is operably linked to the nucleic acid to beexpressed. Within an expression vector, “operably linked” is intended tomean that a nucleic acid of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleic acid(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). Regulatory sequencesinclude promoters, enhancers and other expression control elements(e.g., polyadenylation signals). Regulatory sequences include thosewhich direct constitutive expression of a nucleic acid in many types ofhost cells, those which direct expression of the nucleic acid only incertain host cells (e.g., tissue-specific regulatory sequences), andthose which direct the expression of the nucleic acid upon stimulationwith a particular agent (e.g., inducible regulatory sequences). It willbe appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.The term “host cell” is intended to include a particular subject celltransformed or transfected with a nucleic acid and the progeny orpotential progeny of such a cell. Progeny of such a cell may not beidentical to the parent cell transformed or transfected with the nucleicacid due to mutations or environmental influences that may occur insucceeding generations or integration of the nucleic acid into the hostcell genome.

Expression vectors can be designed for expression of a chimericinfluenza hemagglutinin (HA) polypeptide described herein usingprokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells(using baculovirus expression vectors, see, e.g., Treanor et al., 2007,JAMA, 297(14):1577-1582 incorporated by reference herein in itsentirety), yeast cells, plant cells, algae or mammalian cells). Examplesof yeast host cells include, but are not limited to S. pombe and S.cerevisiae and examples, infra. Examples of mammalian host cellsinclude, but are not limited to, Crucell Per.C6 cells, Vero cells, CHOcells, VERY cells, BHK cells, HeLa cells, COS cells, MDCK cells, 293cells, 3T3 cells or WI38 cells. In certain embodiments, the hosts cellsare myeloma cells, e.g., NS0 cells, 45.6 TG1.7 cells, AF-2 clone 9B5cells, AF-2 clone 9B5 cells, J558L cells, MOPC 315 cells, MPC-11 cells,NCI-H929 cells, NP cells, NS0/1 cells, P3 NS1 Ag4 cells, P3/NS1/1-Ag-4-1cells, P3U1 cells, P3X63Ag8 cells, P3X63Ag8.653 cells, P3X63Ag8U.1cells, RPMI 8226 cells, Sp20-Ag14 cells, U266B1 cells, X63AG8.653 cells,Y3.Ag.1.2.3 cells, and YO cells. Non-limiting examples of insect cellsinclude Sf9, Sf21, Trichoplusia ni, Spodoptera frugiperda and Bombyxmori. In a particular embodiment, a mammalian cell culture system (e.g.Chinese hamster ovary or baby hamster kidney cells) is used forexpression of a chimeric influenza hemagglutinin (HA) polypeptide. Inanother embodiment, a plant cell culture system is used for expressionof a chimeric influenza hemagglutinin (HA) polypeptide. See, e.g., U.S.Pat. Nos. 7,504,560; 6,770,799; 6,551,820; 6,136,320; 6,034,298;5,914,935; 5,612,487; and 5,484,719, and U.S. patent applicationpublication Nos. 2009/0208477, 2009/0082548, 2009/0053762, 2008/0038232,2007/0275014 and 2006/0204487 for plant cells and methods for theproduction of proteins utilizing plant cell culture systems. In specificembodiments, plant cell culture systems are not used for expression of achimeric influenza hemagglutinin (HA) polypeptide described herein.

In certain embodiments, plants (e.g., plants of the genus Nicotiana) maybe engineered to express a chimeric influenza hemagglutinin (HA)polypeptide described herein (e.g., a chimeric influenza hemagglutinin(HA) polypeptide described in Section 5.1). In specific embodiments,plants are engineered to express a chimeric influenza hemagglutinin (HA)polypeptide described herein via an agroinfiltration procedure usingmethods known in the art. For example, nucleic acids encoding a gene ofinterest, e.g., a gene encoding a chimeric influenza hemagglutinin (HA)polypeptide described herein, is introduced into a strain ofAgrobacterium. Subsequently the strain is grown in a liquid culture andthe resulting bacteria are washed and suspended into a buffer solution.The plants are then exposed (e.g., via injection or submersion) to theAgrobacterium that comprises the nucleic acids encoding a fl chimericinfluenza hemagglutinin (HA) polypeptide described herein such that theAgrobacterium transforms the gene of interest to a portion of the plantcells. The chimeric influenza hemagglutinin (HA) polypeptide is thentransiently expressed by the plant and can isolated using methods knownin the art and described herein. (For specific examples see Shoji etal., 2008, Vaccine, 26(23):2930-2934; and D'Aoust et al., 2008, J. PlantBiotechnology, 6(9):930-940). In a specific embodiment, the plant is atobacco plant (e.g., Nicotiana tabacum). In another specific embodiment,the plant is a relative of the tobacco plant (e.g., Nicotianabenthamiana). In another specific embodiment, the chimeric influenzahemagglutinin (HA) polypeptides described herein are expressed in aspecies of soy. In another specific embodiment, the chimeric influenzahemagglutinin (HA) polypeptides described herein are expressed in aspecies of corn. In another specific embodiment, the chimeric influenzahemagglutinin (HA) polypeptides described herein are expressed in aspecies of rice

In other embodiments, algae (e.g., Chlamydomonas reinhardtii) may beengineered to express a chimeric influenza hemagglutinin (HA)polypeptide described herein, e.g., a chimeric influenza hemagglutinin(HA) polypeptide described in Section 5.1 (see, e.g., Rasala et al.,2010, Plant Biotechnology Journal (Published online Mar. 7, 2010)).

In certain embodiments, the plants used to express the chimericinfluenza hemagglutinin (HA) polypeptides described herein areengineered to express components of an N-glycosylation system (e.g., abacterial or mammalian N-glycosylation system), i.e., the plants canperform N-glycosylation.

Plant cells that can be used to express the chimeric influenzahemagglutinin (HA) polypeptides described herein and methods for theproduction of proteins utilizing plant cell culture systems aredescribed in, e.g., U.S. Pat. Nos. 5,929,304; 7,504,560; 6,770,799;6,551,820; 6,136,320; 6,034,298; 5,914,935; 5,612,487; and 5,484,719,U.S. patent application publication Nos. 2009/0208477, 2009/0082548,2009/0053762, 2008/0038232, 2007/0275014 and 2006/0204487, and Shoji etal., 2008, Vaccine, 26(23):2930-2934, and D'Aoust et al., 2008, J. PlantBiotechnology, 6(9):930-940 (which are incorporated herein by referencein their entirety).

The host cells comprising the nucleic acids that encode the chimericinfluenza hemagglutinin (HA) polypeptide described herein can beisolated, e.g., the cells are outside of the body of a subject or areisolated (i.e., separated from) untransfected/untransformed host cells.In certain embodiments, the cells are engineered to express nucleicacids that encode the chimeric influenza hemagglutinin (HA) polypeptidesdescribed herein.

An expression vector can be introduced into host cells via conventionaltransformation or transfection techniques. Such techniques include, butare not limited to, calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, andelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York, andother laboratory manuals. In certain embodiments, a host cell istransiently transfected with an expression vector containing a nucleicacid encoding chimeric influenza hemagglutinin (HA) polypeptide. Inother embodiments, a host cell is stably transfected with an expressionvector containing a nucleic acid encoding a chimeric influenzahemagglutinin (HA) polypeptide.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a nucleic acid thatencodes a selectable marker (e.g., for resistance to antibiotics) isgenerally introduced into the host cells along with the nucleic acid ofinterest. Examples of selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Cellsstably transfected with the introduced nucleic acid can be identified bydrug selection (e.g., cells that have incorporated the selectable markergene will survive, while the other cells die).

As an alternative to recombinant expression of a chimeric influenzahemagglutinin (HA) polypeptide described herein using a host cell, anexpression vector containing a nucleic acid encoding a chimericinfluenza hemagglutinin (HA) polypeptide can be transcribed andtranslated in vitro using, e.g., T7 promoter regulatory sequences and T7polymerase. In a specific embodiment, a coupledtranscription/translation system, such as Promega TNT®, or a cell lysateor cell extract comprising the components necessary for transcriptionand translation may be used to produce a chimeric influenzahemagglutinin (HA) polypeptide described herein.

Once a chimeric influenza hemagglutinin (HA) polypeptide describedherein has been produced, it may be isolated or purified by any methodknown in the art for isolation or purification of a protein, forexample, by chromatography (e.g., ion exchange, affinity, particularlyby affinity for the specific antigen or a “tag” associated with theantigen (e.g., a HIS tag, strep tag/strep II tag, a maltose bindingprotein, a glutatione S-transferase tag, myc tag, HA tag), by Protein A,and chromatography (e.g., sizing column chromatography, hydrophopicinteraction chromatography (HIC), reversed phase chromatography,simulated moving bed chromatography, size eclusion chromatography,monolith chromatography, convective interaction media chromatography,lectin chromatography)), centrifugation, differential solubility,ultrafiltration, precipitation, or by any other standard technique forthe isolation or purification of proteins. In specific embodiments, theisolated/purified chimeric influenza hemagglutinin (HA) polypeptidesdescribed herein are soluble, e.g., made soluble using any method knownto those of skill in the art, e.g., the methods described herein (see,e.g., Section 6.6.1.2).

Provided herein are methods for producing a chimeric influenzahemagglutinin (HA) polypeptide described herein. In one embodiment, themethod comprises culturing a host cell containing a nucleic acidencoding the polypeptide in a suitable medium such that the polypeptideis produced. In some embodiments, the method further comprises isolatingthe polypeptide from the medium or the host cell.

5.4 Influenza Virus Vectors

In one aspect, provided herein are influenza viruses containing achimeric influenza hemagglutinin (HA) polypeptide described herein(e.g., a chimeric influenza hemagglutinin (HA) polypeptide described inSection 5.1). In a specific embodiment, the chimeric influenzahemagglutinin (HA) polypeptide described herein is incorporated into thevirions of the influenza virus. The influenza viruses may be conjugatedto moieties that target the viruses to particular cell types, such asimmune cells. In some embodiments, the virions of the influenza virushave incorporated into them or express a heterologous polypeptide inaddition to a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein. The heterologous polypeptide may be a polypeptide thathas immunopotentiating activity, or that targets the influenza virus toa particular cell type, such as an antibody that binds to an antigen ona specific cell type or a ligand that binds a specific receptor on aspecific cell type.

Influenza viruses containing a chimeric influenza hemagglutinin (HA)polypeptide described herein may be produced by supplying in trans thechimeric influenza hemagglutinin (HA) polypeptide during production ofvirions using techniques known to one skilled in the art, such asreverse genetics and helper-free plasmid rescue. Alternatively, thereplication of a parental influenza virus comprising a genome engineeredto express a chimeric influenza hemagglutinin (HA) polypeptide describedherein in cells susceptible to infection with the virus whereinhemagglutinin function is provided in trans will produce progenyinfluenza viruses containing the chimeric influenza hemagglutinin (HA)polypeptide.

In another aspect, provided herein are influenza viruses comprising agenome engineered to express a chimeric influenza hemagglutinin (HA)polypeptide described herein (e.g., a chimeric influenza hemagglutinin(HA) polypeptide described in Section 5.1). In a specific embodiment,the genome of a parental influenza virus is engineered to encode achimeric influenza hemagglutinin (HA) polypeptide described herein,which is expressed by progeny influenza virus. In another specificembodiment, the genome of a parental influenza virus is engineered toencode a chimeric influenza hemagglutinin (HA) polypeptide describedherein, which is expressed and incorporated into the virions of progenyinfluenza virus. Thus, the progeny influenza virus resulting from thereplication of the parental influenza virus contain a chimeric influenzahemagglutinin (HA) polypeptide described herein. The virions of theparental influenza virus may have incorporated into them a chimericinfluenza hemagglutinin (HA) polypeptide that contains a stem or headdomain from the same or a different type, subtype or strain of influenzavirus. Alternatively, the virions of the parental influenza virus mayhave incorporated into them a moiety that is capable of functionallyreplacing one or more of the activities of influenza virus hemagglutininpolypeptide (e.g., the receptor binding and/or fusogenic activities ofinfluenza virus hemagglutinin). In certain embodiments, one or more ofthe activities of the influenza virus hemagglutinin polypeptide isprovided by a fusion protein comprising (i) an ectodomain of apolypeptide heterologous to influenza virus fused to (ii) atransmembrane domain, or a transmembrane domain and a cytoplasmic domainof an influenza virus hemagglutinin polypeptide. In a specificembodiment, the virions of the parental influenza virus may haveincorporated into them a fusion protein comprising (i) an ectodomain ofa receptor binding/fusogenic polypeptide of an infectious agent otherthan influenza virus fused to (ii) a transmembrane domain, or atransmembrane domain and a cytoplasmic domain of an influenza virushemagglutinin. For a description of fusion proteins that provide one ormore activities of an influenza virus hemagglutinin polypeptide andmethods for the production of influenza viruses engineered to expresssuch fusion proteins, see, e.g., International patent applicationPublication No. WO 2007/064802, published Jun. 7, 2007 and U.S. patentapplication publication no. 2012/0122185; each of which is incorporatedherein by reference in its entirety.

In certain embodiments, the influenza viruses engineered to express oneor more of the chimeric influenza hemagglutinin (HA) polypeptidesdescribed herein comprise a neuraminidase (NA), or fragment thereof,that is from the same source (e.g., influenza virus strain or subtype)as that from which the globular head domain of the chimeric influenzahemagglutinin (HA) polypeptide is derived. In certain embodiments, theinfluenza viruses engineered to express one or more of the chimericinfluenza hemagglutinin polypeptides described herein comprise aneuraminidase (NA), or fragment thereof, that is from the same source(e.g., influenza virus strain or subtype) as that from which theglobular head domain of the chimeric influenza hemagglutinin polypeptideis derived, wherein the globular head domain is heterologous to the stemdomain of the HA1 and/or HA2 subunits of the chimeric influenzahemagglutinin polypeptide. In certain embodiments, the influenza virusesengineered to express one or more of the chimeric influenzahemagglutinin polypeptides described herein comprise a neuraminidase(NA), or fragment thereof, that is from the same source (e.g., influenzavirus strain or subtype) as that from which the HA stem domain of thechimeric influenza hemagglutinin polypeptide is derived.

In some embodiments, the virions of the parental influenza virus haveincorporated into them a heterologous polypeptide. In certainembodiments, the genome of a parental influenza virus is engineered toencode a heterologous polypeptide and a chimeric influenza hemagglutinin(HA) polypeptide, which are expressed by progeny influenza virus. Inspecific embodiments, the chimeric influenza hemagglutinin (HA)polypeptide, the heterologous polypeptide, or both are incorporated intovirions of the progeny influenza virus.

The heterologous polypeptide may be a polypeptide that targets theinfluenza virus to a particular cell type, such as an antibody thatrecognizes an antigen on a specific cell type or a ligand that binds aspecific receptor on a specific cell type. In some embodiments, thetargeting polypeptide replaces the target cell recognition function ofthe virus. In a specific embodiment, the heterologous polypeptidetargets the influenza virus to the same cell types that influenza virusinfects in nature. In other specific embodiments, the heterologouspolypeptide targets the progeny influenza virus to immune cells, such asB cells, T cells, macrophages or dendritic cells. In some embodiments,the heterologous polypeptide recognizes and binds to cell-specificmarkers of antigen presenting cells, such as dendritic cells (e.g., suchas CD44). In one embodiment, the heterologous polypeptide is DC-SIGNwhich targets the virus to dendritic cells. In another embodiment, theheterologous polypeptide is an antibody (e.g., a single-chain antibody)that targets the virus to an immune cell, which may be fused with atransmembrane domain from another polypeptide so that it is incorporatedinto the influenza virus virion. In some embodiments, the antibody is aCD20 antibody, a CD34 antibody, or an antibody against DEC-205.Techniques for engineering viruses to express polypeptides withtargeting functions are known in the art. See, e.g., Yang et al., 2006,PNAS 103: 11479-11484 and United States patent application PublicationNo. 20080019998, published Jan. 24, 2008, and No. 20070020238, publishedJan. 25, 2007, the contents of each of which are incorporated herein intheir entirety.

In another embodiment, the heterologous polypeptide is a viralattachment protein. Non-limiting examples of viruses whose attachmentprotein(s) can be used in this aspect are viruses selected from thegroup of: Lassa fever virus, Hepatitis B virus, Rabies virus, Newcastledisease virus (NDV), a retrovirus such as human immunodeficiency virus,tick-borne encephalitis virus, vaccinia virus, herpesvirus, poliovirus,alphaviruses such as Semliki Forest virus, Ross River virus, and Auravirus (which comprise surface glycoproteins such as E1, E2, and E3),Borna disease virus, Hantaan virus, foamyvirus, and SARS-CoV virus.

In one embodiment, a flavivirus surface glycoprotein may be used, suchas Dengue virus (DV) E protein. In some embodiments, a Sindbis virusglycoprotein from the alphavirus family is used (K. S. Wang, R. J. Kuhn,E. G. Strauss, S. Ou, J. H. Strauss, J. Virol. 66, 4992 (1992)). Incertain embodiments, the heterologous polypeptide is derived from an NDVHN or F protein; a human immunodeficiency virus (HIV) gp160 (or aproduct thereof, such as gp41 or gp120); a hepatitis B virus surfaceantigen (HBsAg); a glycoprotein of herpesvirus (e.g., gD, gE); or VP1 ofpoliovirus.

In another embodiment, the heterologous polypeptide is derived from anynon-viral targeting system known in the art. In certain embodiments, aprotein of a nonviral pathogen such as an intracellular bacteria orprotozoa is used. In some embodiments, the bacterial polypeptide isprovided by, e.g., Chlamydia, Rikettsia, Coxelia, Listeria, Brucella, orLegionella. In some embodiments, protozoan polypeptide is provided by,e.g., Plasmodia species, Leishmania spp., Toxoplasma gondii, orTrypanosoma cruzi. Other exemplary targeting systems are described inWaehler et al., 2007, “Engineering targeted viral vectors for genetherapy,” Nature Reviews Genetics 8: 573-587, which is incorporatedherein in its entirety.

In certain embodiments, the heterologous polypeptide expressed by aninfluenza virus has immunopotentiating (immune stimulating) activity.Non-limiting examples of immunopotentiating polypeptides include, butare not limited to, stimulation molecules, cytokines, chemokines,antibodies and other agents such as Flt-3 ligands. Specific examples ofpolypeptides with immunopotentiating activity include: interferon type1, alpha, beta, or gamma interferon, colony stimulating factors such asgranulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin(IL)-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23,tumor necrosis factor (TNF)-β, TNFα, B7.1, B7.2, 4-1BB, CD40 ligand(CD40L), and drug-inducible CD40 (iCD40) (see, e.g., Hanks, B. A., etal. 2005. Nat Med 11:130-137, which is incorporated herein by referencein its entirety.)

Since the genome of influenza A and B viruses consist of eight (8)single-stranded, negative sense segments (influenza C viruses consist ofseven (7) single-stranded, negative sense segments), the genome of aparental influenza virus may be engineered to express a chimericinfluenza hemagglutinin (HA) polypeptide described herein (and any otherpolypeptide, such as a heterologous polypeptide) using a recombinantsegment and techniques known to one skilled in the art, such a reversegenetics and helper-free plasmid rescue. In one embodiment, therecombinant segment comprises a nucleic acid encoding the chimericinfluenza hemagglutinin (HA) polypeptide as well as the 3′ and 5′incorporation signals which are required for proper replication,transcription and packaging of the vRNAs (Fujii et al., 2003, Proc.Natl. Acad. Sci. USA 100:2002-2007; Zheng, et al., 1996, Virology217:242-251, both of which are incorporated by reference herein in theirentireties). In a specific embodiment, the recombinant segment uses the3′ and 5′ noncoding and/or nontranslated sequences of segments ofinfluenza viruses that are from a different or the same type, subtype orstrain as the parental influenza virus. In some embodiments, therecombinant segment comprises the 3′ noncoding region of an influenzavirus hemagglutinin polypeptide, the untranslated regions of aninfluenza virus hemagglutinin polypeptide, and the 5′ non-coding regionof an influenza virus hemagglutinin polypeptide. In specificembodiments, the recombinant segment comprises the 3′ and 5′ noncodingand/or nontranslated sequences of the HA segment of an influenza virusthat is the same type, subtype or strain as the influenza virus type,subtype or strain as the HA1 N-terminal stem segment, the HA1 C-terminalstem segment, the globular head domain, and/or the HA2 of a chimericinfluenza hemagglutinin (HA) polypeptide. In certain embodiments, therecombinant segment encoding the chimeric influenza hemagglutinin (HA)polypeptide may replace the HA segment of a parental influenza virus. Insome embodiments, the recombinant segment encoding the chimericinfluenza hemagglutinin (HA) polypeptide may replace the NS1 gene of theparental influenza virus. In some embodiments, the recombinant segmentencoding the chimeric influenza hemagglutinin (HA) polypeptide mayreplace the NA gene of the parental influenza virus. Exemplary influenzavirus strains that can be used to express the chimeric influenzahemagglutinin (HA) polypeptides described herein include Ann Arbor/1/50,A/Ann Arbor/6/60, A/Puerto Rico/8/34, A/South Dakota/6/2007,A/Uruguay/716/2007, A/California/07/2009, A/Perth/16/2009,A/Brisbane/59/2007, A/Brisbane/10/2007, A/Leningrad/134/47/57,B/Brisbane/60/2008, B/Yamagata/1/88, A/Panama/2007/99, A/Wyoming/3/03,and A/WSN/33.

In some embodiments, an influenza virus hemagglutinin gene segmentencodes a chimeric influenza hemagglutinin (HA) polypeptide describedherein. In specific embodiments, the influenza virus hemagglutinin genesegment and at least one other influenza virus gene segment comprisepackaging signals that enable the influenza virus hemagglutinin genesegment and the at least one other gene segment to segregate togetherduring replication of a recombinant influenza virus (see, Gao & Palese2009, PNAS 106:15891-15896; International Application Publication No.WO11/014645; and U.S. Patent Application Publication No. 2012/0244183).

In some embodiments, the genome of a parental influenza virus may beengineered to express a chimeric influenza hemagglutinin (HA)polypeptide described herein using a recombinant segment that isbicistronic. Bicistronic techniques allow the engineering of codingsequences of multiple proteins into a single mRNA through the use ofinternal ribosome entry site (IRES) sequences. IRES sequences direct theinternal recruitment of ribosomes to the RNA molecule and allowdownstream translation in a cap independent manner. Briefly, a codingregion of one protein is inserted into the open reading frame (ORF) of asecond protein. The insertion is flanked by an IRES and any untranslatedsignal sequences necessary for proper expression and/or function. Theinsertion must not disrupt the ORF, polyadenylation or transcriptionalpromoters of the second protein (see, e.g., Garcia-Sastre et al., 1994,J. Virol. 68:6254-6261 and Garcia-Sastre et al., 1994 Dev. Biol. Stand.82:237-246, each of which is hereby incorporated by reference in itsentirety). See also, e.g., U.S. Pat. No. 6,887,699, U.S. Pat. No.6,001,634, U.S. Pat. No. 5,854,037 and U.S. Pat. No. 5,820,871, each ofwhich is incorporated herein by reference in its entirety. Any IRESknown in the art or described herein may be used in accordance with theinvention (e.g., the IRES of BiP gene, nucleotides 372 to 592 of GenBankdatabase entry HUMGRP78; or the IRES of encephalomyocarditis virus(EMCV), nucleotides 1430-2115 of GenBank database entry CQ867238). Thus,in certain embodiments, a parental influenza virus is engineered tocontain a bicistronic RNA segment that expresses the chimeric influenzahemagglutinin (HA) polypeptide and another polypeptide, such as a geneexpressed by the parental influenza virus. In some embodiments, theparental influenza virus gene is the HA gene. In some embodiments, theparental influenza virus gene is the NA gene. In some embodiments, theparental influenza virus gene is the NS1 gene.

Techniques known to one skilled in the art may be used to produce aninfluenza virus containing a chimeric influenza hemagglutinin (HA)polypeptide described herein and an influenza virus comprising a genomeengineered to express a chimeric influenza hemagglutinin (HA)polypeptide described herein. For example, reverse genetics techniquesmay be used to generate such an influenza virus. Briefly, reversegenetics techniques generally involve the preparation of syntheticrecombinant viral RNAs that contain the non-coding regions of thenegative-strand, viral RNA which are essential for the recognition byviral polymerases and for packaging signals necessary to generate amature virion. The recombinant RNAs are synthesized from a recombinantDNA template and reconstituted in vitro with purified viral polymerasecomplex to form recombinant ribonucleoproteins (RNPs) which can be usedto transfect cells. A more efficient transfection is achieved if theviral polymerase proteins are present during transcription of thesynthetic RNAs either in vitro or in vivo. The synthetic recombinantRNPs can be rescued into infectious virus particles. The foregoingtechniques are described in U.S. Pat. No. 5,166,057 issued Nov. 24,1992; in U.S. Pat. No. 5,854,037 issued Dec. 29, 1998; in EuropeanPatent Publication EP 0702085A1, published Feb. 20, 1996; in U.S. patentapplication Ser. No. 09/152,845; in International Patent PublicationsPCT WO 97/12032 published Apr. 3, 1997; WO 96/34625 published Nov. 7,1996; in European Patent Publication EP A780475; WO 99/02657 publishedJan. 21, 1999; WO 98/53078 published Nov. 26, 1998; WO 98/02530published Jan. 22, 1998; WO 99/15672 published Apr. 1, 1999; WO 98/13501published Apr. 2, 1998; WO 97/06270 published Feb. 20, 1997; and EPO 780475A1 published Jun. 25, 1997, each of which is incorporated byreference herein in its entirety.

Alternatively, helper-free plasmid technology may be used to produce aninfluenza virus containing chimeric influenza hemagglutinin (HA)polypeptide described herein and an influenza virus comprising a genomeengineered to express a chimeric influenza hemagglutinin (HA)polypeptide described herein. Briefly, full length cDNAs of viralsegments are amplified using PCR with primers that include uniquerestriction sites, which allow the insertion of the PCR product into theplasmid vector (Flandorfer et al., 2003, J. Virol. 77:9116-9123; Nakayaet al., 2001, J. Virol. 75:11868-11873; both of which are incorporatedherein by reference in their entireties). The plasmid vector is designedso that an exact negative (vRNA sense) transcript is expressed. Forexample, the plasmid vector may be designed to position the PCR productbetween a truncated human RNA polymerase I promoter and a hepatitisdelta virus ribozyme sequence such that an exact negative (vRNA sense)transcript is produced from the polymerase I promoter. Separate plasmidvectors comprising each viral segment as well as expression vectorscomprising necessary viral proteins may be transfected into cellsleading to production of recombinant viral particles. In anotherexample, plasmid vectors from which both the viral genomic RNA and mRNAencoding the necessary viral proteins are expressed may be used. For adetailed description of helper-free plasmid technology see, e.g.,International Publication No. WO 01/04333; U.S. Pat. Nos. 6,951,754,7,384,774, 6,649,372, and 7,312,064; Fodor et al., 1999, J. Virol.73:9679-9682; Quinlivan et al., 2005, J. Virol. 79:8431-8439; Hoffmannet al., 2000, Proc. Natl. Acad. Sci. USA 97:6108-6113; Neumann et al.,1999, Proc. Natl. Acad. Sci. USA 96:9345-9350; Enami and Enami, 2000, J.Virol. 74(12):5556-5561; and Pleschka et al., 1996, J. Virol.70(6):4188-4192, which are incorporated herein by reference in theirentireties.

The influenza viruses described herein may be propagated in anysubstrate that allows the virus to grow to titers that permit their usein accordance with the methods described herein. In one embodiment, thesubstrate allows the viruses to grow to titers comparable to thosedetermined for the corresponding wild-type viruses. In certainembodiments, the substrate is one which is biologically relevant to theinfluenza virus or to the virus from which the HA function is derived.In a specific embodiment, an attenuated influenza virus by virtue of,e.g., a mutation in the NS1 gene, may be propagated in an IFN-deficientsubstrate. For example, a suitable IFN-deficient substrate may be onethat is defective in its ability to produce or respond to interferon, oris one which an IFN-deficient substrate may be used for the growth ofany number of viruses which may require interferon-deficient growthenvironment. See, for example, U.S. Pat. No. 6,573,079, issued Jun. 3,2003, U.S. Pat. No. 6,852,522, issued Feb. 8, 2005, and U.S. Pat. No.7,494,808, issued Feb. 24, 2009, the entire contents of each of which isincorporated herein by reference in its entirety. In a specificembodiment, the virus is propagated in embryonated eggs (e.g., chickeneggs). In a specific embodiment, the virus is propagated in 8 day old,9-day old, 8-10 day old, 10 day old, 11-day old, 10-12 day old, or12-day old embryonated eggs (e.g., chicken eggs). In certainembodiments, the virus is propagated in MDCK cells, Vero cells, 293Tcells, or other cell lines known in the art. In certain embodiments, thevirus is propagated in cells derived from embryonated eggs.

The influenza viruses described herein may be isolated and purified byany method known to those of skill in the art. In one embodiment, thevirus is removed from cell culture and separated from cellularcomponents, typically by well known clarification procedures, e.g., suchas gradient centrifugation and column chromatography, and may be furtherpurified as desired using procedures well known to those skilled in theart, e.g., plaque assays.

In certain embodiments, the influenza viruses, or influenza viruspolypeptides, genes or genome segments for use as described herein areobtained or derived from an influenza A virus. In certain embodiments,the influenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from two ormore influenza A virus subtypes or strains.

In some embodiments, the influenza viruses, or influenza viruspolypeptides, genes or genome segments for use as described herein areobtained or derived from an influenza B virus. In certain embodiments,the influenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from two ormore influenza B virus subtypes or strains. In other embodiments, theinfluenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from acombination of influenza A and influenza B virus subtypes or strains.

In some embodiments, the influenza viruses, or influenza viruspolypeptides, genes or genome segments for use as described herein areobtained or derived from an influenza C virus. In certain embodiments,the influenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from two ormore influenza C virus subtypes or strains. In other embodiments, theinfluenza viruses, or influenza virus polypeptides, genes or genomesegments for use as described herein are obtained or derived from acombination of influenza C virus and influenza A virus and/or influenzaB virus subtypes or strains.

In certain embodiments, the influenza viruses provided herein have anattenuated phenotype. In specific embodiments, the attenuated influenzavirus is based on influenza A virus. In other embodiments, theattenuated influenza virus is based on influenza B virus. In yet otherembodiments, the attenuated influenza virus is based on influenza Cvirus. In other embodiments, the attenuated influenza virus may comprisegenes or genome segments from one or more strains or subtypes ofinfluenza A, influenza B, and/or influenza C virus. In some embodiments,the attenuated backbone virus comprises genes from an influenza A virusand an influenza B virus.

In specific embodiments, attenuation of influenza virus is desired suchthat the virus remains, at least partially, infectious and can replicatein vivo, but only generate low titers resulting in subclinical levels ofinfection that are non-pathogenic. Such attenuated viruses areespecially suited for embodiments described herein wherein the virus oran immunogenic composition thereof is administered to a subject toinduce an immune response. Attenuation of the influenza virus can beaccomplished according to any method known in the art, such as, e.g.,selecting viral mutants generated by chemical mutagenesis, mutation ofthe genome by genetic engineering, selecting reassortant viruses thatcontain segments with attenuated function, or selecting for conditionalvirus mutants (e.g., cold-adapted viruses). Alternatively, naturallyoccurring attenuated influenza viruses may be used as influenza virusbackbones for the influenza virus vectors.

In one embodiment, an influenza virus may be attenuated, at least inpart, by virtue of substituting the HA gene of the parental influenzavirus with a chimeric influenza hemagglutinin (HA) polypeptide describedherein. In some embodiments, an influenza virus may be attenuated, atleast in part, by engineering the influenza virus to express a mutatedNS1 gene that impairs the ability of the virus to antagonize thecellular interferon (IFN) response. Examples of the types of mutationsthat can be introduced into the influenza virus NS1 gene includedeletions, substitutions, insertions and combinations thereof. One ormore mutations can be introduced anywhere throughout the NS1 gene (e.g.,the N-terminus, the C-terminus or somewhere in between) and/or theregulatory element of the NS1 gene. In one embodiment, an attenuatedinfluenza virus comprises a genome having a mutation in an influenzavirus NS1 gene resulting in a deletion consisting of 5, preferably 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 99, 100,105, 110, 115, 120, 125, 126, 130, 135, 140, 145, 150, 155, 160, 165,170 or 175 amino acid residues from the C-terminus of NS1, or a deletionof between 5-170, 25-170, 50-170, 100-170, 100-160, or 105-160 aminoacid residues from the C-terminus. In another embodiment, an attenuatedinfluenza virus comprises a genome having a mutation in an influenzavirus NS1 gene such that it encodes an NS1 protein of amino acidresidues 1-130, amino acid residues 1-126, amino acid residues 1-120,amino acid residues 1-115, amino acid residues 1-110, amino acidresidues 1-100, amino acid residues 1-99, amino acid residues 1-95,amino acid residues 1-85, amino acid residues 1-83, amino acid residues1-80, amino acid residues 1-75, amino acid residues 1-73, amino acidresidues 1-70, amino acid residues 1-65, or amino acid residues 1-60,wherein the N-terminus amino acid is number 1. For examples of NS1mutations and influenza viruses comprising a mutated NS1, see, e.g.,U.S. Pat. Nos. 6,468,544 and 6,669,943; and Li et al., 1999, J. Infect.Dis. 179:1132-1138, each of which is incorporated by reference herein inits entirety.

In another embodiment, an influenza virus may be attenuated, at least inpart, by mutating an NA or M gene of the virus as described in theliterature.

5.5 Non-Influenza Virus Vectors

In one aspect, provided herein are non-influenza viruses containing achimeric influenza hemagglutinin (HA) polypeptide described herein(e.g., a chimeric influenza hemagglutinin (HA) polypeptide described inSection 5.1). In a specific embodiment, the chimeric influenzahemagglutinin (HA) polypeptide described herein is incorporated into thevirions of the non-influenza virus. In a specific embodiment, thechimeric influenza hemagglutinin (HA) polypeptide described herein iscontained in/expressed by a purified (e.g., plaque purified) or isolatedvirus. The non-influenza viruses may be conjugated to moieties thattarget the viruses to particular cell types, such as immune cells. Insome embodiments, the virions of the non-influenza virus haveincorporated into them or express a heterologous polypeptide in additionto a chimeric influenza hemagglutinin (HA) polypeptide described herein.The heterologous polypeptide may be a polypeptide that hasimmunopotentiating activity, or that targets the non-influenza virus toa particular cell type, such as an antibody that recognizes an antigenon a specific cell type or a ligand that binds a specific receptor on aspecific cell type. See Section 5.4 supra for examples of suchheterologous polypeptides.

Non-influenza viruses containing/expressing a chimeric influenzahemagglutinin (HA) polypeptide described herein can be produced usingtechniques known to those skilled in the art. Non-influenza virusescontaining a chimeric influenza hemagglutinin (HA) polypeptide describedherein may be produced by supplying in trans the chimeric influenzahemagglutinin (HA) polypeptide during production of virions usingtechniques known to one skilled in the art. Alternatively, thereplication of a parental non-influenza virus comprising a genomeengineered to express a chimeric influenza hemagglutinin (HA)polypeptide described herein in cells susceptible to infection with thevirus wherein hemagglutinin function is provided in trans will produceprogeny viruses containing the chimeric influenza hemagglutinin (HA)polypeptide.

Any virus type, subtype or strain including, but not limited to,naturally occurring strains, variants or mutants, mutagenized viruses,reassortants and/or genetically modified viruses may be used as anon-influenza virus vector. In a specific embodiment, the parentalnon-influenza virus is not a naturally occurring virus. In anotherspecific embodiment, the parental non-influenza virus is a geneticallyengineered virus. In certain embodiments, an enveloped virus ispreferred for the expression of a membrane bound chimeric influenzahemagglutinin (HA) polypeptide described herein.

In an exemplary embodiment, the non-influenza virus vector is aNewcastle disease virus (NDV). In another embodiment, the non-influenzavirus vector is a vaccinia virus. In another embodiment, thenon-influenza virus vector is a baculovirus. In other exemplary,non-limiting, embodiments, the non-influenza virus vector is adenovirus,adeno-associated virus (AAV), hepatitis B virus, retrovirus (such as,e.g., a gammaretrovirus such as Mouse Stem Cell Virus (MSCV) genome orMurine Leukemia Virus (MLV), e.g., Moloney murine leukemia virus,oncoretrovirus, or lentivirus), an alphavirus (e.g., Venezuelan equineencephalitis virus), a rhabdovirus (such as vesicular stomatitis virus(VSV) or papillomaviruses), poxvirus (such as, e.g., vaccinia virus, aMVA-T7 vector, or fowlpox), metapneumovirus, measles virus, herpesvirus(such as herpes simplex virus), or foamyvirus. See, e.g., Lawrie andTumin, 1993, Cur. Opin. Genet. Develop. 3, 102-109 (retroviral vectors);Bett et al., 1993, J. Virol. 67, 5911 (adenoviral vectors); Zhou et al.,1994, J. Exp. Med. 179, 1867 (adeno-associated virus vectors); Dubenskyet al., 1996, J. Virol. 70, 508-519 (viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses and viral vectorsfrom the alpha virus genus such as those derived from Sindbis andSemliki Forest Viruses); U.S. Pat. No. 5,643,576 (Venezuelan equineencephalitis virus); WO 96/34625 (VSV); Ohe et al., 1995, Human GeneTherapy 6, 325-333; Woo et al., WO 94/12629; Xiao & Brandsma, 1996,Nucleic Acids. Res. 24, 2630-2622 (papillomaviruses); and Bukreyev andCollins, 2008, Curr Opin Mol Ther. 10:46-55 (NDV), each of which isincorporated by reference herein in its entirety.

In a specific embodiment, the non-influenza virus vector is NDV. Any NDVtype, subtype or strain may serve as the backbone that is engineered toexpress a chimeric influenza hemagglutinin (HA) polypeptide describedherein, including, but not limited to, naturally-occurring strains,variants or mutants, mutagenized viruses, reassortants and/orgenetically engineered viruses. In a specific embodiment, the NDV thatserves as the backbone for genetic engineering is a naturally-occurringstrain. In certain embodiments, the NDV that serves as the backbone forgenetic engineering is a lytic strain. In other embodiments, the NDVthat serves as the backbone for genetic engineering is a non-lyticstrain. In certain embodiments, the NDV that serves as the backbone forgenetic engineering is lentogenic strain. In some embodiments, the NDVthat serves as the backbone for genetic engineering is a mesogenicstrain. In other embodiments, the NDV that serves as the backbone forgenetic engineering is a velogenic strain. Specific examples of NDVstrains include, but are not limited to, the 73-T strain, Ulster strain,MTH-68 strain, Italien strain, Hickman strain, PV701 strain, Hitchner B1strain, La Sota strain, YG97 strain, MET95 strain, and F48E9 strain. Ina specific embodiment, the NDV that serves as the backbone for geneticengineering is the Hitchner B1 strain. In another specific embodiment,the NDV that serves as the backbone for genetic engineering is the LaSota strain.

In one embodiment, the NDV used as the backbone for a non-influenzavirus vector is engineered to express a modified F protein in which thecleavage site of the F protein is replaced with one containing one ortwo extra arginine residues, allowing the mutant cleavage site to beactivated by ubiquitously expressed proteases of the furin family.Specific examples of NDVs that express such a modified F proteininclude, but are not limited to, rNDV/F2aa and rNDV/F3aa. For adescription of mutations introduced into a NDV F protein to produce amodified F protein with a mutated cleavage site, see, e.g., Park et al.(2006) “Engineered viral vaccine constructs with dual specificity: Avianinfluenza and Newcastle disease.” PNAS USA 103: 8203-2808, which isincorporated herein by reference in its entirety.

In one embodiment, the non-influenza virus vector is a poxvirus. Apoxvirus vector may be based on any member of the poxyiridae, inparticular, a vaccinia virus or an avipox virus (e.g., such ascanarypox, fowlpox, etc.) that provides suitable sequences for vaccinevectors. In a specific embodiment, the poxviral vector is a vacciniavirus vector. Suitable vaccinia viruses include, but are not limited to,the Copenhagen (VC-2) strain (Goebel, et al., Virol 179: 247-266, 1990;Johnson, et al., Virol. 196: 381-401, 1993), modified Copenhagen strain(NYVAC) (U.S. Pat. No. 6,265,189), the WYETH strain and the modifiedAnkara (MVA) strain (Antoine, et al., Virol. 244: 365-396, 1998). Othersuitable poxviruses include fowlpox strains such as ALVAC and TROVACvectors that provide desirable properties and are highly attenuated(see, e.g., U.S. Pat. No. 6,265,189; Tartaglia et al., In AIDS ResearchReviews, Koff, et al., eds., Vol. 3, Marcel Dekker, N.Y., 1993; andTartaglia et al., 1990, Reviews in Immunology 10: 13-30, 1990).

Methods of engineering non-influenza viruses to express influenzapolypeptides are well known in the art, as are methods for attenuating,propagating, and isolating and purifying such viruses. For suchtechniques with respect to NDV vectors, see, e.g., InternationalPublication No. WO 01/04333; U.S. Pat. Nos. 7,442,379, 6,146,642,6,649,372, 6,544,785 and 7,384,774; Swayne et al. (2003). Avian Dis.47:1047-1050; and Swayne et al. (2001). J. Virol. 11868-11873, each ofwhich is incorporated by reference in its entirety. For such techniqueswith respect to poxviruses, see, e.g., Piccini, et al., Methods ofEnzymology 153: 545-563, 1987; International Publication No. WO96/11279; U.S. Pat. No. 4,769,330; U.S. Pat. No. 4,722,848; U.S. Pat.No. 4,769,330; U.S. Pat. No. 4,603,112; U.S. Pat. No. 5,110,587; U.S.Pat. No. 5,174,993; EP 83 286; EP 206 920; Mayr et al., Infection 3:6-14, 1975; and Sutter and Moss, Proc. Natl. Acad. Sci. USA 89:10847-10851, 1992. In certain embodiments, the non-influenza virus isattenuated.

Exemplary considerations for the selection of a non-influenza virusvector, particularly for use in compositions for administration to asubject, are safety, low toxicity, stability, cell type specificity, andimmunogenicity, particularly, antigenicity of the chimeric influenzahemagglutinin (HA) polypeptide described herein expressed by thenon-influenza virus vector.

5.6 Virus-Like Particles and Virosomes

The chimeric influenza hemagglutinin (HA) polypeptides described herein(e.g., the chimeric influenza hemagglutinin (HA) polypeptides describedin Section 5.1) can be incorporated into virus-like particle (VLP)vectors, e.g., purified/isolated VLPs. VLPs generally comprise a viralpolypeptide(s) typically derived from a structural protein(s) of avirus. In some embodiments, the VLPs are not capable of replicating. Incertain embodiments, the VLPs may lack the complete genome of a virus orcomprise a portion of the genome of a virus. In some embodiments, theVLPs are not capable of infecting a cell. In some embodiments, the VLPsexpress on their surface one or more of viral (e.g., virus surfaceglycoprotein) or non-viral (e.g., antibody or protein) targetingmoieties known to one skilled in the art or described herein. In someembodiments, the VLPs comprise a chimeric influenza hemagglutinin (HA)polypeptide described herein and a viral structural protein, such as HIVgag. In a specific embodiment, the VLPs comprise a chimeric influenzahemagglutinin (HA) polypeptide described herein and an HIV gagpolypeptide.

Methods for producing and characterizing recombinantly produced VLPshave been described based on several viruses, including influenza virus(Bright et al. (2007) Vaccine. 25:3871), human papilloma virus type 1(Hagnesee et al. (1991) J. Virol. 67:315), human papilloma virus type 16(Kirnbauer et al. Proc. Natl. Acad. Sci. (1992) 89:12180), HIV-1 (Hafferet al., (1990) J. Virol. 64:2653), and hepatitis A (Winokur (1991)65:5029), each of which is incorporated herein in its entirety. Methodsfor expressing VLPs that contain NDV proteins are provided by Pantua etal. (2006) J. Virol. 80:11062-11073, and in United States patentapplication Publication No. 20090068221, published Mar. 12, 2009, eachof which is incorporated in its entirety herein. In a specificembodiment, the VLPs comprising chimeric influenza hemagglutinin (HA)polypeptides described herein are generated using baculovirus. In otherembodiments, the VLPs comprising chimeric influenza hemagglutinin (HA)polypeptide described herein are generated using 293T cells.

In specific embodiments, VLPs, e.g., VLPs comprising a chimericinfluenza hemagglutinin (HA) polypeptide described herein, are expressedin cells (e.g., 293T cells). In certain embodiments, the VLPs areexpressed in cells that express surface glycoproteins that comprisesialic acid. In accordance with such embodiments, the cells are culturedin the presence of neuraminidase (e.g., viral of bacterialneuraminidase). In certain embodiments, VLPs, e.g., VLPs comprising achimeric influenza hemagglutinin (HA) polypeptide described herein, areexpressed in cells that do not express surface glycoproteins thatcomprise sialic acid.

In a specific embodiment, a chimeric influenza hemagglutinin (HA)polypeptide described herein may be incorporated into a virosome. Avirosome containing a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein may be produced using techniques known to those skilledin the art. For example, a virosome may be produced by disrupting apurified virus, extracting the genome, and reassembling particles withthe viral proteins (e.g., a chimeric influenza hemagglutinin (HA)polypeptide described herein) and lipids to form lipid particlescontaining viral proteins.

5.7 Bacterial Vectors

In a specific embodiment, bacteria may be engineered to express achimeric influenza hemagglutinin (HA) polypeptide described herein(e.g., a chimeric influenza hemagglutinin (HA) polypeptide described inSection 5.1). Suitable bacteria for expression of a chimeric influenzahemagglutinin (HA) polypeptide described herein include, but are notlimited to, Listeria, Salmonella, Shigella sp., Mycobacteriumtuberculosis, E. coli, Neisseria meningitides, Brucella abortus,Brucella melitensis, Borrelia burgdorferi, Lactobacillus, Campylobacter,Lactococcus, Bifidobacterium, and Francisella tularensis. In a specificembodiment, the bacteria engineered to express a chimeric influenzahemagglutinin (HA) polypeptide described herein are attenuated.Techniques for the production of bacteria engineered to express aheterologous polypeptide are known in the art and can be applied to theexpression of a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein. See, e.g., United States Patent ApplicationPublication No. 20080248066, published Oct. 9, 2008, and United StatesPatent Application Publication No. 20070207171, published Sep. 6, 2007,each of which are incorporated by reference herein in their entirety. Incertain embodiments, the bacterial vectors used herein possess theability to perform N-linked glycosylation, e.g., such bacteria naturallypossess N-glycosylation machinery (e.g., Campylobacter) or have beengenetically engineered to possess N-glycosylation machinery.

5.8 Generation of Antibodies Against Chimeric Influenza Hemagglutinin(HA) Polypeptides

The chimeric influenza hemagglutinin (HA) polypeptides described herein(e.g., the chimeric influenza hemagglutinin (HA) polypeptides describedin Section 5.1), nucleic acids encoding such polypeptides, or vectorscomprising such nucleic acids or polypeptides described herein may beused to elicit neutralizing antibodies against influenza, for example,against the stalk region of an influenza virus hemagglutininpolypeptide. In a specific embodiment, the chimeric influenzahemagglutinin (HA) polypeptides, nucleic acids encoding suchpolypeptides, or vectors comprising such nucleic acids or polypeptidesdescribed herein may be administered to a non-human subject (e.g., amouse, rabbit, rat, guinea pig, etc.) to induce an immune response thatincludes the production of antibodies which may be isolated usingtechniques known to one of skill in the art (e.g., immunoaffinitychromatography, centrifugation, precipitation, etc.).

In certain embodiments, human antibodies directed against the chimericinfluenza hemagglutinin (HA) polypeptides described herein can begenerated using non-human subjects (e.g., transgenic mice) that arecapable of producing human antibodies. For example, human antibodies canbe produced using transgenic mice which are incapable of expressingfunctional endogenous immunoglobulins, but which can express humanimmunoglobulin genes. For example, the human heavy and light chainimmunoglobulin gene complexes may be introduced randomly or byhomologous recombination into mouse embryonic stem cells. Alternatively,the human variable region, constant region, and diversity region may beintroduced into mouse embryonic stem cells in addition to the humanheavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. For an overviewof this technology for producing human antibodies, see Lonberg andHuszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entirety. Companies such as Abgenix, Inc. (Freemont, Calif.),Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.) can beengaged to provide human antibodies directed against a selected antigen.In addition, non-human subjects may be transplanted with humanperipheral blood leukocytes, splenocytes, or bone marrow (e.g., TriomaTechniques XTL) so that human antibodies that bind to a chimericinfluenza hemagglutinin (HA) polypeptide described herein are generated.

Alternatively, the chimeric influenza hemagglutinin (HA) polypeptidesdescribed herein may be used to screen for antibodies from antibodylibraries. For example, an isolated chimeric influenza hemagglutinin(HA) polypeptide may be immobilized to a solid support (e.g., a silicagel, a resin, a derivatized plastic film, a glass bead, cotton, aplastic bead, a polystyrene bead, an alumina gel, or a polysaccharide, amagnetic bead), and screened for binding to antibodies. As analternative, the antibodies may be immobilized to a solid support andscreened for binding to the isolated chimeric influenza hemagglutinin(HA) polypeptide. Any screening assay, such as a panning assay, ELISA,surface plasmon resonance, or other antibody screening assay known inthe art may be used to screen for antibodies that bind to the chimericinfluenza hemagglutinin (HA) polypeptide. The antibody library screenedmay be a commercially available antibody library, an in vitro generatedlibrary, or a library obtained by identifying and cloning or isolatingantibodies from an individual infected with influenza. In particularembodiments, the antibody library is generated from a survivor of aninfluenza virus outbreak. Antibody libraries may be generated inaccordance with methods known in the art. In a particular embodiment,the antibody library is generated by cloning the antibodies and usingthem in phage display libraries or a phagemid display library.

Antibodies identified in the methods described herein may be tested forneutralizing activity and lack of autoreactivity using the biologicalassays known in the art or described herein. In one embodiment, anantibody isolated from a non-human animal or an antibody libraryneutralizes a hemagglutinin polypeptide from more than one influenzasubtype. In some embodiments, an antibody elicited or identified using achimeric influenza hemagglutinin (HA) polypeptide described herein, anucleic acid encoding such a polypeptide, or a vector encoding such anucleic acid or polypeptide neutralizes an influenza H3 virus. In someembodiments, an antibody elicited or identified using a chimericinfluenza hemagglutinin (HA) polypeptide described herein, a nucleicacid encoding such a polypeptide, or a vector comprising such a nucleicacid or polypeptide neutralizes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or 16 or more subtypes or strains of influenza virus. In oneembodiment, the neutralizing antibody neutralizes one or more influenzaA viruses and one or more influenza B viruses. In particularembodiments, the neutralizing antibody is not, or does not bind the sameepitope as CR6261, CR6325, CR6329, CR6307, CR6323, 2A, D7, D8, F10, G17,H40, A66, D80, E88, E90, H98, C179 (produced by hybridoma FERM BP-4517;clones sold by Takara Bio, Inc. (Otsu, Shiga, Japan)), and/or AI3C (FERMBP-4516); or any other antibody described in Ekiert D C et al. (2009)Antibody Recognition of a Highly Conserved Influenza Virus Epitope.Science (published in Science Express Feb. 26, 2009); Kashyap et al.(2008) Combinatorial antibody libraries from survivors of the TurkishH5N1 avian influenza outbreak reveal virus neutralization strategies.Proc Natl Acad Sci USA 105: 5986-5991; Sui et al. (2009) Structural andfunctional bases for broad-spectrum neutralization of avian and humaninfluenza A viruses. Nat Struct Mol Biol 16: 265-273; U.S. Pat. Nos.5,589,174, 5,631,350, 6,337,070, and 6,720,409; InternationalApplication No. PCT/US2007/068983 published as International PublicationNo. WO 2007/134237; International Application No. PCT/US2008/075998published as International Publication No. WO 2009/036157; InternationalApplication No. PCT/EP2007/059356 published as International PublicationNo. WO 2008/028946; and International Application No. PCT/US2008/085876published as International Publication No. WO 2009/079259. In otherembodiments, the neutralizing antibody is not an antibody described inWang et al. (2010) “Broadly Protective Monoclonal Antibodies against H3Influenza Viruses following Sequential Immunization with DifferentHemagglutinins,” PLOS Pathogens 6(2):1-9. In particular embodiments, theneutralizing antibody does not use the Ig VH1-69 segment. In someembodiments, the interaction of the neutralizing antibody with theantigen is not mediated exclusively by the heavy chain.

Antibodies identified or elicited using a chimeric influenzahemagglutinin (HA) polypeptide described herein, a nucleic acid encodingsuch a polypeptide, or a vector comprising such a nucleic acid orpolypeptide include immunoglobulin molecules and immunologically activeportions of immunoglobulin molecules, i.e., molecules that contain anantigen binding site that specifically binds to a hemagglutininpolypeptide. The immunoglobulin molecules may be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass of immunoglobulin molecule. Antibodies include,but are not limited to, monoclonal antibodies, multispecific antibodies,human antibodies, humanized antibodies, chimeric antibodies,single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab′)fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies elicitedor identified using a method described herein), and epitope-bindingfragments of any of the above.

Antibodies elicited or identified using a chimeric influenzahemagglutinin (HA) polypeptide described herein, nucleic acids encodingsuch a polypeptide or a vector comprising such a nucleic acid orpolypeptide may be used in diagnostic immunoassays, passiveimmunotherapy, and generation of antiidiotypic antibodies. Theantibodies before being used in passive immunotherapy may be modified,e.g., the antibodies may be chimerized or humanized. See, e.g., U.S.Pat. Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741, each of which is incorporated herein byreference in its entirety, for reviews on the generation of chimeric andhumanized antibodies. In addition, the ability of the antibodies toneutralize hemagglutinin polypeptides and the specificity of theantibodies for the polypeptides may be tested prior to using theantibodies in passive immunotherapy.

Antibodies elicited or identified using a chimeric influenzahemagglutinin (HA) polypeptide described herein, a nucleic acid encodingsuch a polypeptide, or a vector comprising such a nucleic acid orpolypeptide may be used to monitor the efficacy of a therapy and/ordisease progression. Any immunoassay system known in the art may be usedfor this purpose including, but not limited to, competitive andnoncompetitive assay systems using techniques such as radioimmunoassays,ELISA (enzyme linked immunosorbent assays), “sandwich” immunoassays,precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays and immunoelectrophoresis assays, to name but a few.

Antibodies elicited or identified using a chimeric influenzahemagglutinin (HA) polypeptide described herein, a nucleic acid encodingsuch a polypeptide, or a vector comprising such a nucleic acid orpolypeptide may be used in the production of antiidiotypic antibody. Theantiidiotypic antibody can then in turn be used for immunization, inorder to produce a subpopulation of antibodies that bind a particularantigen of influenza, e.g., a neutralizing epitope of a hemagglutininpolypeptide (Jerne, 1974, Ann. Immunol. (Paris) 125c:373; Jerne et al.,1982, EMBO J. 1:234, incorporated herein by reference in its entirety).

5.9 Stimulation of Cells Chimeric Influenza Hemagglutinin (HA)Polypeptides

In another aspect, provided herein are methods for stimulating cells exvivo with a chimeric influenza hemagglutinin (HA) polypeptide describedherein (e.g., a chimeric influenza hemagglutinin (HA) polypeptidedescribed in Section 5.1). Such cells, e.g., dendritic cells, may beused in vitro to generate antibodies against the chimeric influenzahemagglutinin (HA) polypeptide or may themselves be administered to asubject by, e.g., an adoptive transfer technique known in the art. See,e.g., United States patent application Publication No. 20080019998,published Jan. 24, 2008, which is incorporated herein by reference inits entirety, for a description of adoptive transfer techniques. Incertain embodiments, when cells that have been stimulated ex vivo with achimeric influenza hemagglutinin (HA) polypeptide described herein areadministered to a subject, the cells are not mammalian cells (e.g., CB-1cells).

In one non-limiting example, a vector, e.g., an influenza virus vector,engineered to express a chimeric influenza hemagglutinin (HA)polypeptide described herein can be used to generate dendritic cells(DCs) that express the chimeric influenza hemagglutinin (HA) polypeptideand display immunostimulatory properties directed against an influenzavirus hemagglutinin polypeptide. Such DCs may be used to expand memory Tcells and are potent stimulators of T cells, including chimericinfluenza hemagglutinin (HA) polypeptide-specific cytotoxic T lymphocyteclones. See Strobel et al., 2000, Human Gene Therapy 11:2207-2218, whichis incorporated herein by reference in its entirety.

A chimeric influenza hemagglutinin (HA) polypeptide described herein maybe delivered to a target cell in any way that allows the polypeptide tocontact the target cell, e.g., a DC, and deliver the polypeptide to thetarget cell. In certain embodiments, the chimeric influenzahemagglutinin (HA) polypeptide described herein is delivered to asubject, as described herein. In some such embodiments, cells contactedwith the polypeptide may be isolated and propagated.

In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide described herein is delivered to a target cell in vitro.Techniques known to one of skill in the art may be used to deliver thepolypeptide to target cells. For example, target cells may be contactedwith the polypeptide in a tissue culture plate, tube or other container.The polypeptide may be suspended in media and added to the wells of aculture plate, tube or other container. The media containing thepolypeptide may be added prior to plating of the cells or after thecells have been plated. The target cells are preferably incubated withthe polypeptide for a sufficient amount of time to allow the polypeptideto contact the cells. In certain embodiments, the cells are incubatedwith the polypeptide for about 1 hour or more, about 5 hours or more,about 10 hours or more, about 12 hours or more, about 16 hours or more,about 24, hours or more, about 48 hours or more, about 1 hour to about12 hours, about 3 hours to about 6 hours, about 6 hours to about 12hours, about 12 hours to about 24 hours, or about 24 hours to about 48hours. In certain embodiments, wherein the chimeric influenzahemagglutinin (HA) polypeptide is in a virus, the contacting of thetarget cells comprises infecting the cells with the virus.

The target cells may be from any species, including, e.g., humans, mice,rats, rabbits and guinea pigs. In some embodiments, target cells are DCsobtained from a healthy subject or a subject in need of treatment. Incertain embodiments, target cells are DCs obtained from a subject inwhom it is desired to stimulate an immune response to the polypeptide.Methods of obtaining cells from a subject are well known in the art.

5.10 Compositions

The nucleic acids, vectors, polypeptides, bacteria, antibodies, and/orcells described herein (sometimes referred to herein as “activecompounds”) may be incorporated into compositions. In a specificembodiment, the compositions are pharmaceutical compositions, such asimmunogenic compositions (e.g., vaccine formulations). Thepharmaceutical compositions provided herein can be in any form thatallows for the composition to be administered to a subject. In aspecific embodiment, the pharmaceutical compositions are suitable forveterinary and/or human administration. The compositions may be used inmethods of preventing or treating an influenza virus disease.

In one embodiment, a pharmaceutical composition comprises a chimericinfluenza hemagglutinin (HA) polypeptide described herein (e.g., achimeric influenza hemagglutinin (HA) polypeptide described in Section5.1), in an admixture with a pharmaceutically acceptable carrier. Inanother embodiment, a pharmaceutical composition comprises a nucleicacid encoding a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein (e.g., a chimeric influenza hemagglutinin (HA)polypeptide described in Section 5.1), in an admixture with apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition comprises an expression vector comprising anucleic acid encoding a chimeric influenza hemagglutinin (HA)polypeptide described herein (e.g., a chimeric influenza hemagglutinin(HA) polypeptide described in Section 5.1), in an admixture with apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition comprises an influenza virus or non-influenzavirus containing a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein (e.g., a chimeric influenza hemagglutinin (HA)polypeptide described in Section 5.1), in an admixture with apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition comprises an influenza virus or non-influenzavirus having a genome engineered to express a chimeric influenzahemagglutinin (HA) polypeptide described herein (e.g., a chimericinfluenza hemagglutinin (HA) polypeptide described in Section 5.1), inadmixture with a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical composition comprises a virus-like particleor virosome containing a chimeric influenza hemagglutinin (HA)polypeptide described herein (e.g., a chimeric influenza hemagglutinin(HA) polypeptide described in Section 5.1), in an admixture with apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition comprises a bacteria expressing or engineeredto express a chimeric influenza hemagglutinin (HA) polypeptide describedherein (e.g., a chimeric influenza hemagglutinin (HA) polypeptidedescribed in Section 5.1), in an admixture with a pharmaceuticallyacceptable carrier. In another embodiment, a pharmaceutical compositioncomprises cells stimulated with a chimeric influenza hemagglutinin (HA)polypeptide described herein (e.g., a chimeric influenza hemagglutinin(HA) polypeptide described in Section 5.1), in an admixture with apharmaceutically acceptable carrier.

In some embodiments, a pharmaceutical composition may comprise one ormore other therapies in addition to a therapy that utilizes a chimericinfluenza hemagglutinin (HA) polypeptide described herein.

As used herein, the term “pharmaceutically acceptable” means approved bya regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeiae foruse in animals, and more particularly in humans. The term “carrier”refers to a diluent, adjuvant, excipient, or vehicle with which thepharmaceutical composition is administered. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable excipients includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Examples of suitable pharmaceutical carriers are describedin “Remington's Pharmaceutical Sciences” by E. W. Martin. Theformulation should suit the mode of administration.

In a specific embodiment, pharmaceutical compositions are formulated tobe suitable for the intended route of administration to a subject. Forexample, the pharmaceutical composition may be formulated to be suitablefor parenteral, oral, intradermal, transdermal, colorectal,intraperitoneal, and rectal administration. In a specific embodiment,the pharmaceutical composition may be formulated for intravenous, oral,intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular,topical, intradermal, transdermal or pulmonary administration.

In certain embodiments, biodegradable polymers, such as ethylene vinylacetate, polyanhydrides, polyethylene glycol (PEGylation), polymethylmethacrylate polymers, polylactides, poly(lactide-co-glycolides),polyglycolic acid, collagen, polyorthoesters, and polylactic acid, maybe used as carriers. In some embodiments, the active compounds areprepared with carriers that increase the protection of the compoundagainst rapid elimination from the body, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Methods for preparation of such formulations will be apparent to thoseskilled in the art. Liposomes or micelles can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. In certain embodiments, the pharmaceuticalcompositions comprise one or more adjuvants.

In specific embodiments, the immunogenic compositions described hereinare monovalent formulations, e.g., monovalent formulations comprising acH5/1 chimeric influenza hemagglutinin polypeptide described herein, acH5/3 chimeric influenza hemagglutinin polypeptide described herein, acH7/3 chimeric influenza hemagglutinin polypeptide described herein, acH5/B chimeric influenza hemagglutinin polypeptide described herein, acH7/B chimeric influenza hemagglutinin polypeptide described herein, ora cHB/B chimeric influenza hemagglutinin polypeptide described herein.

In other specific embodiments, the immunogenic compositions describedherein are multivalent formulations, eg., bivalent and trivalentformulations. In one example, a multivalent formulation comprises morethan one vector expressing a chimeric influenza hemagglutinin (HA)polypeptide described herein. In certain embodiments, a multivalentformulation may comprise one or more different chimeric influenzahemagglutinin (HA) polypeptides described herein expressed using asingle vector. In specific embodiments, a bivalent vaccine formulationprovided herein may comprise a combination of a cH5/1 chimeric influenzahemagglutinin polypeptide described herein and a cH5/3 chimericinfluenza hemagglutinin polypeptide described herein; or a combinationof a cH5/1 chimeric influenza hemagglutinin polypeptide described hereinand a cH7/3 chimeric influenza hemagglutinin polypeptide describedherein. In specific embodiments, a trivalent vaccine formulationprovided herein may comprise (i) a combination of a cH5/1 chimericinfluenza hemagglutinin polypeptide described herein and a cH5/3chimeric influenza hemagglutinin polypeptide described herein and eitherof a cH5/B, a cH7/B, or a cB/B chimeric influenza hemagglutininpolypeptide described herein; or (ii) a combination of a cH5/1 chimericinfluenza hemagglutinin polypeptide described herein and a cH7/3chimeric influenza hemagglutinin polypeptide described herein and eitherof a cH5/B, a cH7/B, or a cB/B chimeric influenza hemagglutininpolypeptide described herein.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise a preservative, e.g., the mercury derivativethimerosal. In a specific embodiment, the pharmaceutical compositionsdescribed herein comprises 0.001% to 0.01% thimerosal. In otherembodiments, the pharmaceutical compositions described herein do notcomprise a preservative. In a specific embodiment, thimerosal is usedduring the manufacture of a pharmaceutical composition described hereinand the thimerosal is removed via purification steps followingproduction of the pharmaceutical composition, i.e., the pharmaceuticalcomposition contains trace amounts of thimerosal (<0.3 μg of mercury perdose after purification; such pharmaceutical compositions are consideredthimerosal-free products).

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise egg protein (e.g., ovalbumin or other eggproteins). The amount of egg protein in the pharmaceutical compositionsdescribed herein may range from about 0.0005 to about 1.2. μg of eggprotein to 1 ml of pharmaceutical composition. In other embodiments, thepharmaceutical compositions described herein do not comprise eggprotein.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise one or more antimicrobial agents (e.g.,antibiotics) including, but not limited to gentamicin, neomycin,polymyxin (e.g., polymyxin B), and kanamycin, streptomycin. In otherembodiments, the pharmaceutical compositions described herein do notcomprise any antibiotics.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise one or more components used to inactivate a virus,e.g., formalin or formaldehyde or a detergent such as sodiumdeoxycholate, octoxynol 9 (Triton X-100), and octoxynol 10. In otherembodiments, the pharmaceutical compositions described herein do notcomprise any components used to inactivate a virus.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise gelatin. In other embodiments, the pharmaceuticalcompositions described herein do not comprise gelatin.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise one or more buffers, e.g., phosphate buffer andsucrose phosphate glutamate buffer. In other embodiments, thepharmaceutical compositions described herein do not comprise buffers.

In certain embodiments, the pharmaceutical compositions described hereinadditionally comprise one or more salts, e.g., sodium chloride, calciumchloride, sodium phosphate, monosodium glutamate, and aluminum salts(e.g., aluminum hydroxide, aluminum phosphate, alum (potassium aluminumsulfate), or a mixture of such aluminum salts). In other embodiments,the pharmaceutical compositions described herein do not comprise salts.

In specific embodiments, the pharmaceutical compositions describedherein are low-additive influenza virus vaccines, i.e., thepharmaceutical compositions do not comprise one or more additivescommonly found in influenza virus vaccines. Low-additive influenzavaccines have been described (see, e.g., International Application No.PCT/IB2008/002238 published as International Publication No. WO09/001,217 which is herein incorporated by reference in its entirety).

The pharmaceutical compositions described herein can be included in acontainer, pack, or dispenser together with instructions foradministration.

The pharmaceutical compositions described herein can be stored beforeuse, e.g., the pharmaceutical compositions can be stored frozen (e.g.,at about −20° C. or at about −70° C.); stored in refrigerated conditions(e.g., at about 4° C.); or stored at room temperature (see InternationalApplication No. PCT/IB2007/001149 published as International PublicationNo. WO 07/110,776, which is herein incorporated by reference in itsentirety, for methods of storing compositions comprising influenzavaccines without refrigeration).

In certain embodiments, when the active compound in a pharmaceuticalcomposition described herein is a cell engineered to express a chimericinfluenza hemagglutinin (HA) polypeptide described herein, the cells inthe pharmaceutical composition are not mammalian cells (e.g., CB-1cells).

5.10.1 Subunit Vaccines

In a specific embodiment, provided herein are subunit vaccinescomprising a chimeric influenza hemagglutinin (HA) polypeptide describedherein. In some embodiments, a subunit vaccine comprises a chimericinfluenza hemagglutinin (HA) polypeptide described herein and one ormore surface glycoproteins (e.g., influenza virus neuraminidase), othertargeting moieties, or adjuvants. In specific embodiments, a subunitvaccine comprises a single chimeric influenza hemagglutinin (HA)polypeptide described herein. In other embodiments, a subunit vaccinecomprises two, three, four or more chimeric influenza hemagglutinin (HA)polypeptides described herein. In specific embodiments, the chimericinfluenza hemagglutinin (HA) polypeptides used in a subunit vaccine arenot membrane-bound, i.e., are soluble.

The subunit vaccines provided herein comprise an effective amount ofchimeric influenza hemagglutinin (HA) polypeptide. In certainembodiments, the subunit vaccines provided herein comprise about 5, 6,7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 μg ofone or more chimeric influenza hemagglutinin (HA) polypeptides describedherein. In certain embodiments, the subunit vaccines provided hereincomprise about 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80,80-90, 90-100, 100-110, 110-120, 120-130, 130-140, or 140-150 μg of oneor more chimeric influenza hemagglutinin (HA) polypeptides describedherein.

In a specific embodiment, a monovalent subunit vaccine provided hereincomprises between 7.5 μg to 90 μg of a chimeric influenza hemagglutinin(HA) polypeptide described herein. In another specific embodiment, abivalent subunit vaccine provided herein comprises between 7.5 μg to 90μg of a first chimeric influenza hemagglutinin (HA) polypeptidedescribed herein and between 7.5 μg to 90 μg of a second chimericinfluenza hemagglutinin (HA) polypeptide described herein. In anotherspecific embodiment, a trivalent subunit vaccine provided hereincomprises between 7.5 μg to 90 μg of a first chimeric influenzahemagglutinin (HA) polypeptide described herein, between 7.5 μg to 90 μgof a second chimeric influenza hemagglutinin (HA) polypeptide describedherein, and between 7.5 μg to 90 μg of a third chimeric influenzahemagglutinin (HA) polypeptide described herein.

In certain embodiments, provided herein are subunit vaccines comprisingabout 10 μg to about 60 μg of one or more chimeric influenzahemagglutinin (HA) polypeptides described herein, about 0.001% to 0.01%thimerosal, about 0.1 μg to about 1.0 μg chicken egg protein, about 1.0μg to about 5.0 μg polymyxin, about 1.0 μg to about 5.0 μg neomycin,about 0.1 μg to about 0.5 μg betapropiolactone, and about 0.001 to about0.05% w/v of nonylphenol ethoxylate per dose.

In a specific embodiment, a subunit vaccine provided herein comprises orconsists of a 0.5 ml dose that comprises 45 μg of a chimeric influenzahemagglutinin (HA) polypeptide described herein, ≦1.0 μg of mercury(from thimerosal), ≦1.0 μg chicken egg protein (i.e., ovalbumin), ≦3.75μg polymyxin, and ≦2.5 μg neomycin. In some embodiments, a subunitvaccine provided herein additionally comprises or consists of not morethan 0.5 μg betapropiolactone, and not more than 0.015% w/v ofnonylphenol ethoxylate per dose. In some embodiments, the 0.5 ml dosesubunit vaccine is packaged in a pre-filled syringe.

In a specific embodiment, a subunit vaccine provided herein consists ofa 5.0 ml multidose vial (0.5 ml per dose) that comprises 45 μg of achimeric influenza hemagglutinin (HA) polypeptide described herein, 25.0μg of mercury (from thimerosal), ≦1.0 μg chicken egg protein (i.e.,ovalbumin), ≦3.75 μg polymyxin, and ≦2.5 μg neomycin. In someembodiments, a subunit vaccine provided herein additionally comprises orconsists of not more than 0.5 μg betapropiolactone, and not more than0.015% w/v of nonylphenol ethoxylate per dose.

In a specific embodiment, the subunit vaccine is prepared usinginfluenza virus that was propagated in embryonated chicken eggs (i.e.,the components of the subunit vaccine (e.g., a chimeric influenzahemagglutinin (HA) polypeptide described herein) are isolated from virusthat was propagated in embryonated chicken eggs). In another specificembodiment, the subunit vaccine is prepared using influenza virus thatwas not propagated in embryonated chicken eggs (i.e., the components ofthe subunit vaccine (e.g., a chimeric influenza hemagglutinin (HA)polypeptide described herein) are isolated from virus that was notpropagated in embryonated chicken eggs). In another specific embodiment,the subunit vaccine is prepared using influenza virus that waspropagated in mammalian cells, e.g., immortalized human cells (see,e.g., International Application No. PCT/EP2006/067566 published asInternational Publication No. WO 07/045,674 which is herein incorporatedby reference in its entirety) or canine kidney cells such as MDCK cells(see, e.g., International Application No. PCT/IB2007/003536 published asInternational Publication No. WO 08/032,219 which is herein incorporatedby reference in its entirety) (i.e., the components of the subunitvaccine (e.g., a chimeric influenza hemagglutinin (HA) polypeptide) areisolated from virus that was propagated in mammalian cells). In anotherspecific embodiment, the chimeric influenza hemagglutinin (HA)polypeptides in the subunit vaccines provided herein are prepared usingan expression vector, e.g., a viral vector, plant vector or a bacterialvector (i.e., the chimeric influenza hemagglutinin (HA) polypeptide inthe subunit vaccine are obtained/isolated from an expression vector).

In a specific embodiment, provided herein is a monovalent subunitvaccine comprising a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein. In a specific embodiment, provided herein is amonovalent subunit vaccine comprising a cH5/1 chimeric influenzahemagglutinin polypeptide (e.g., a cH5/1 chimeric influenzahemagglutinin polypeptide described in Section 5.1). In another specificembodiment, provided herein is a monovalent subunit vaccine comprising acH5/3 chimeric influenza hemagglutinin polypeptide (e.g., a cH5/3chimeric influenza hemagglutinin polypeptide described in Section 5.1).In another specific embodiment, provided herein is a monovalent subunitvaccine comprising a cH7/3 chimeric influenza hemagglutinin polypeptide(e.g., a cH7/3 chimeric influenza hemagglutinin polypeptide described inSection 5.1). In another specific embodiment, provided herein is amonovalent subunit vaccine comprising a cH5/B chimeric influenzahemagglutinin polypeptide (e.g., a cH5/B chimeric influenzahemagglutinin polypeptide described in Section 5.1). In another specificembodiment, provided herein is a monovalent subunit vaccine comprising acH7/B chimeric influenza hemagglutinin polypeptide (e.g., a cH7/1chimeric influenza hemagglutinin polypeptide described in Section 5.1).In another specific embodiment, provided herein is a monovalent subunitvaccine comprising a cB/B chimeric influenza hemagglutinin polypeptide(e.g., a B/B chimeric influenza hemagglutinin polypeptide described inSection 5.1).

In specific embodiments, provided herein are bivalent subunit vaccinescomprising a chimeric influenza hemagglutinin (HA) polypeptide describedherein. In a specific embodiment, provided herein is a bivalent subunitvaccine comprising a cH5/1 chimeric influenza hemagglutinin polypeptideand a cH5/3 chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a bivalent subunit vaccinecomprising a cH5/1 chimeric influenza hemagglutinin polypeptide and acH7/3 chimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a bivalent subunit vaccine comprising acH5/1 chimeric influenza hemagglutinin polypeptide and a cH5/B chimericinfluenza hemagglutinin polypeptide. In another specific embodiment,provided herein is a bivalent subunit vaccine comprising a cH5/1chimeric influenza hemagglutinin polypeptide and a cH7/B chimericinfluenza hemagglutinin polypeptide. In another specific embodiment,provided herein is a bivalent subunit vaccine comprising a cH5/1chimeric influenza hemagglutinin polypeptide and a cB/B chimericinfluenza hemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalent subunitvaccine comprising a cH5/3 chimeric influenza hemagglutinin polypeptideand a cH7/3 chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a bivalent subunit vaccinecomprising a cH5/3 chimeric influenza hemagglutinin polypeptide and acH5/B chimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a bivalent subunit vaccine comprising acH5/3 chimeric influenza hemagglutinin polypeptide and a cH7/B chimericinfluenza hemagglutinin polypeptide. In another specific embodiment,provided herein is a bivalent subunit vaccine comprising a cH5/3chimeric influenza hemagglutinin polypeptide and a cB/B chimericinfluenza hemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalent subunitvaccine comprising a cH7/3 chimeric influenza hemagglutinin polypeptideand a cH5/B chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a bivalent subunit vaccinecomprising a cH7/3 chimeric influenza hemagglutinin polypeptide and acH7/B chimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a bivalent subunit vaccine comprising acH7/3 chimeric influenza hemagglutinin polypeptide and a cB/B chimericinfluenza hemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalent subunitvaccine comprising a cH5/B chimeric influenza hemagglutinin polypeptideand a cH7/B chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a bivalent subunit vaccinecomprising a cH5/B chimeric influenza hemagglutinin polypeptide and acB/B chimeric influenza hemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalent subunitvaccine comprising a cH7/B chimeric influenza hemagglutinin polypeptideand a cB/B chimeric influenza hemagglutinin polypeptide.

In specific embodiments, provided herein are trivalent subunit vaccinescomprising viruses that contain and/or comprise a genome that encodes achimeric influenza hemagglutinin (HA) polypeptide described herein. In aspecific embodiment, provided herein is a trivalent subunit vaccinecomprising a cH5/1 chimeric influenza hemagglutinin polypeptide, a cH5/3chimeric influenza hemagglutinin polypeptide, and a cH5/B chimericinfluenza hemagglutinin polypeptide. In another specific embodiment,provided herein is a trivalent subunit vaccine comprising a cH5/1chimeric influenza hemagglutinin polypeptide, a cH5/3 chimeric influenzahemagglutinin polypeptide, and a cH7/B chimeric influenza hemagglutininpolypeptide. In another specific embodiment, provided herein is atrivalent subunit vaccine comprising a cH5/1 chimeric influenzahemagglutinin polypeptide, a cH5/3 chimeric influenza hemagglutininpolypeptide, and a cB/B chimeric influenza hemagglutinin polypeptide.

In another specific embodiment, provided herein is a trivalent subunitvaccine comprising a cH5/1 chimeric influenza hemagglutinin polypeptide,a cH7/3 chimeric influenza hemagglutinin polypeptide, and a cH5/Bchimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a trivalent subunit vaccine comprising acH5/1 chimeric influenza hemagglutinin polypeptide, a cH7/3 chimericinfluenza hemagglutinin polypeptide, and a cH7/B chimeric influenzahemagglutinin polypeptide. In another specific embodiment, providedherein is a trivalent subunit vaccine comprising a cH5/1 chimericinfluenza hemagglutinin polypeptide, a cH7/3 chimeric influenzahemagglutinin polypeptide, and a cB/B chimeric influenza hemagglutininpolypeptide.

In a specific embodiment, a subunit vaccine provided herein does notcomprise a cH5/3 chimeric influenza hemagglutinin polypeptide comprisingthe globular head domain of A/Vietnam/1203/2004 (H5). In anotherspecific embodiment, a subunit vaccine provided herein does not comprisecH5/3 chimeric influenza hemagglutinin polypeptide comprising the stemdomain of A/Perth/16/2009 (H3).

In a specific embodiment, a subunit vaccine provided herein does notcomprise a cH7/3 chimeric influenza hemagglutinin polypeptide comprisingthe globular head domain of A/mallard/Alberta/24/2001 (H7). In anotherspecific embodiment, a subunit vaccine provided herein does not comprisea cH7/3 chimeric influenza hemagglutinin polypeptide comprising the stemdomain of A/Perth/16/2009 (H3).

5.10.2 Live Virus Vaccines

In one embodiment, provided herein are immunogenic compositions (e.g.,vaccines) comprising live virus containing a chimeric influenzahemagglutinin (HA) polypeptide described herein. In another embodiment,provided herein are immunogenic compositions (e.g., vaccines) comprisinglive virus that is engineered to encode a chimeric influenzahemagglutinin (HA) polypeptide described herein, which is expressed byprogeny virus produced in the subjects administered the compositions. Inspecific embodiments, the chimeric influenza hemagglutinin (HA)polypeptide is membrane-bound. In other specific embodiments, thechimeric influenza hemagglutinin (HA) polypeptide is not membrane-bound,i.e., it is soluble. In particular embodiments, the live virus is aninfluenza virus, such as described in Section 5.4, supra. In otherembodiments, the live virus is a non-influenza virus, such as describedin Section 5.5, supra. In some embodiments, the live virus isattenuated. In some embodiments, an immunogenic composition comprisestwo, three, four or more live viruses containing or engineered toexpress two, three, four or more different chimeric influenzahemagglutinin (HA) polypeptide described herein.

In certain embodiments, provided herein are immunogenic compositions(e.g., vaccines) comprising about 10⁵ to about 10¹⁰ fluorescent focusunits (FFU) of live attenuated influenza virus containing one or morechimeric influenza hemagglutinin (HA) polypeptide described herein,about 0.1 to about 0.5 mg monosodium glutamate, about 1.0 to about 5.0mg hydrolyzed procine gelatin, about 1.0 to about 5.0 mg arginine, about10 to about 15 mg sucrose, about 1.0 to about 5.0 mg dibasic potassiumphosphate, about 0.5 to about 2.0 mg monobasic potassium phosphate, andabout 0.001 to about 0.05 μg/ml gentamicin sulfate per dose. In someembodiments, the immunogenic compositions (e.g., vaccines) are packagedas pre-filled sprayers containing single 0.2 ml doses.

In a specific embodiment, provided herein are immunogenic compositions(e.g., vaccines) comprising 10^(6.5) to 10^(7.5) FFU of live attenuatedinfluenza virus containing one or more chimeric influenza hemagglutinin(HA) polypeptide described herein, 0.188 mg monosodium glutamate, 2.0 mghydrolyzed procine gelatin, 2.42 mg arginine, 13.68 mg sucrose, 2.26 mgdibasic potassium phosphate, 0.96 mg monobasic potassium phosphate, and<0.015 μg/ml gentamicin sulfate per dose. In some embodiments, theimmunogenic compositions (e.g., vaccines) are packaged as pre-filledsprayers containing single 0.2 ml doses.

In a specific embodiment, the live virus that contains a chimericinfluenza hemagglutinin (HA) polypeptide described herein is propagatedin embryonated chicken eggs before its use in an immunogenic compositiondescribed herein. In another specific embodiment, the live virus thatcontains a chimeric influenza hemagglutinin (HA) polypeptide describedherein is not propagated in embryonated chicken eggs before its use inan immunogenic composition described herein. In another specificembodiment, the live virus that contains a chimeric influenzahemagglutinin (HA) polypeptide described herein is propagated inmammalian cells, e.g., immortalized human cells (see, e.g.,International Application No. PCT/EP2006/067566 published asInternational Publication No. WO 07/045,674 which is herein incorporatedby reference in its entirety) or canine kidney cells such as MDCK cells(see, e.g., International Application No. PCT/IB2007/003536 published asInternational Publication No. WO 08/032,219 which is herein incorporatedby reference in its entirety) before its use in an immunogeniccomposition described herein.

An immunogenic composition comprising a live virus for administration toa subject may be preferred because multiplication of the virus in thesubject may lead to a prolonged stimulus of similar kind and magnitudeto that occurring in natural infections, and therefore, confersubstantial, long lasting immunity.

In a specific embodiment, provided herein is a monovalent live virusvaccine comprising a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein. In a specific embodiment, provided herein is amonovalent live virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH5/1 chimeric influenza hemagglutininpolypeptide (e.g., a cH5/1 chimeric influenza hemagglutinin polypeptidedescribed in Section 5.1). In another specific embodiment, providedherein is a monovalent live virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/3 chimericinfluenza hemagglutinin polypeptide (e.g., a cH5/3 chimeric influenzahemagglutinin polypeptide described in Section 5.1). In another specificembodiment, provided herein is a live virus vaccine comprising a virusthat contains and/or comprises a genome that encodes a cH7/3 chimericinfluenza hemagglutinin polypeptide (e.g., a cH7/3 chimeric influenzahemagglutinin polypeptide described in Section 5.1). In another specificembodiment, provided herein is a monovalent live virus vaccinecomprising a virus that contains and/or comprises a genome that encodesa cH5/B chimeric influenza hemagglutinin polypeptide (e.g., a cH5/Bchimeric influenza hemagglutinin polypeptide described in Section 5.1).In another specific embodiment, provided herein is a monovalent livevirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH7/B chimeric influenza hemagglutinin polypeptide (e.g.,a cH7/1 chimeric influenza hemagglutinin polypeptide described inSection 5.1). In another specific embodiment, provided herein is amonovalent live virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cB/B chimeric influenza hemagglutininpolypeptide (e.g., a B/B chimeric influenza hemagglutinin polypeptidedescribed in Section 5.1).

In specific embodiments, provided herein are bivalent live virusvaccines comprising viruses that contain and/or comprise a genome thatencodes a chimeric influenza hemagglutinin (HA) polypeptide describedherein. In a specific embodiment, provided herein is a bivalent livevirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH5/1 chimeric influenza hemagglutinin polypeptide and avirus that contains and/or comprises a genome that encodes a cH5/3chimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a bivalent live virus vaccine comprisinga virus that contains and/or comprises a genome that encodes a cH5/1chimeric influenza hemagglutinin polypeptide and a virus that containsand/or comprises a genome that encodes a cH7/3 chimeric influenzahemagglutinin polypeptide. In another specific embodiment, providedherein is a bivalent live virus vaccine comprising a virus that containsand/or comprises a genome that encodes a cH5/1 chimeric influenzahemagglutinin polypeptide and a virus that contains and/or comprises agenome that encodes a cH5/B chimeric influenza hemagglutininpolypeptide. In another specific embodiment, provided herein is abivalent live virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a virus that contains and/or comprises agenome that encodes a cH5/1 chimeric influenza hemagglutinin polypeptideand a virus that contains and/or comprises a genome that encodes a cH7/Bchimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a bivalent live virus vaccine comprisinga virus that contains and/or comprises a genome that encodes a cH5/1chimeric influenza hemagglutinin polypeptide and a virus that containsand/or comprises a genome that encodes a cB/B chimeric influenzahemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalent live virusvaccine comprising a virus that contains and/or comprises a genome thatencodes a cH5/3 chimeric influenza hemagglutinin polypeptide and a virusthat contains and/or comprises a genome that encodes a cH7/3 chimericinfluenza hemagglutinin polypeptide. In another specific embodiment,provided herein is a bivalent live virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/3 chimericinfluenza hemagglutinin polypeptide and a virus that contains and/orcomprises a genome that encodes a cH5/B chimeric influenza hemagglutininpolypeptide. In another specific embodiment, provided herein is abivalent live virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH5/3 chimeric influenza hemagglutininpolypeptide and a virus that contains and/or comprises a genome thatencodes a cH7/B chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a bivalent live virus vaccinecomprising a virus that contains and/or comprises a genome that encodesa cH5/3 chimeric influenza hemagglutinin polypeptide and a virus thatcontains and/or comprises a genome that encodes a cB/B chimericinfluenza hemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalent live virusvaccine comprising a virus that contains and/or comprises a genome thatencodes a cH7/3 chimeric influenza hemagglutinin polypeptide and a virusthat contains and/or comprises a genome that encodes a cH5/B chimericinfluenza hemagglutinin polypeptide. In another specific embodiment,provided herein is a bivalent live virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH7/3 chimericinfluenza hemagglutinin polypeptide and a virus that contains and/orcomprises a genome that encodes a cH7/B chimeric influenza hemagglutininpolypeptide. In another specific embodiment, provided herein is abivalent live virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH7/3 chimeric influenza hemagglutininpolypeptide and a virus that contains and/or comprises a genome thatencodes a cB/B chimeric influenza hemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalent live virusvaccine comprising a virus that contains and/or comprises a genome thatencodes a cH5/B chimeric influenza hemagglutinin polypeptide and a virusthat contains and/or comprises a genome that encodes a cH7/B chimericinfluenza hemagglutinin polypeptide. In another specific embodiment,provided herein is a bivalent live virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/B chimericinfluenza hemagglutinin polypeptide and a virus that contains and/orcomprises a genome that encodes a cB/B chimeric influenza hemagglutininpolypeptide.

In another specific embodiment, provided herein is a bivalent live virusvaccine comprising a virus that contains and/or comprises a genome thatencodes a cH7/B chimeric influenza hemagglutinin polypeptide and a virusthat contains and/or comprises a genome that encodes a cB/B chimericinfluenza hemagglutinin polypeptide.

In specific embodiments, provided herein are trivalent live virusvaccines comprising viruses that contain and/or comprise a genome thatencodes a chimeric influenza hemagglutinin (HA) polypeptide describedherein. In a specific embodiment, provided herein is a trivalent livevirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH5/1 chimeric influenza hemagglutinin polypeptide, avirus that contains and/or comprises a genome that encodes a cH5/3chimeric influenza hemagglutinin polypeptide, and a virus that containsand/or comprises a genome that encodes a cH5/B chimeric influenzahemagglutinin polypeptide. In another specific embodiment, providedherein is a trivalent live virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/1 chimericinfluenza hemagglutinin polypeptide, a virus that contains and/orcomprises a genome that encodes a cH5/3 chimeric influenza hemagglutininpolypeptide, and a virus that contains and/or comprises a genome thatencodes a cH7/B chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a trivalent live virus vaccinecomprising a virus that contains and/or comprises a genome that encodesa cH5/1 chimeric influenza hemagglutinin polypeptide, a virus thatcontains and/or comprises a genome that encodes a cH5/3 chimericinfluenza hemagglutinin polypeptide, and a virus that contains and/orcomprises a genome that encodes a cB/B chimeric influenza hemagglutininpolypeptide.

In another specific embodiment, provided herein is a trivalent livevirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH5/1 chimeric influenza hemagglutinin polypeptide, avirus that contains and/or comprises a genome that encodes a cH7/3chimeric influenza hemagglutinin polypeptide, and a virus that containsand/or comprises a genome that encodes a cH5/B chimeric influenzahemagglutinin polypeptide. In another specific embodiment, providedherein is a trivalent live virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/1 chimericinfluenza hemagglutinin polypeptide, a virus that contains and/orcomprises a genome that encodes a cH7/3 chimeric influenza hemagglutininpolypeptide, and a virus that contains and/or comprises a genome thatencodes a cH7/B chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a trivalent live virus vaccinecomprising a virus that contains and/or comprises a genome that encodesa cH5/1 chimeric influenza hemagglutinin polypeptide, a virus thatcontains and/or comprises a genome that encodes a cH7/3 chimericinfluenza hemagglutinin polypeptide, and a virus that contains and/orcomprises a genome that encodes a cB/B chimeric influenza hemagglutininpolypeptide.

In a specific embodiment, a live virus vaccine provided herein does notcomprise a virus that contains and/or comprises a genome that encodes acH5/3 chimeric influenza hemagglutinin polypeptide comprising theglobular head domain of A/Vietnam/1203/2004 (H5). In another specificembodiment, a live virus vaccine provided herein does not comprise avirus that contains and/or comprises a genome that encodes a cH5/3chimeric influenza hemagglutinin polypeptide comprising the stem domainof A/Perth/16/2009 (H3).

In a specific embodiment, a live virus vaccine provided herein does notcomprise a virus that contains and/or comprises a genome that encodes acH7/3 chimeric influenza hemagglutinin polypeptide comprising theglobular head domain of A/mallard/Alberta/24/2001 (H7). In anotherspecific embodiment, a live virus vaccine provided herein does notcomprise a virus that contains and/or comprises a genome that encodes acH7/3 chimeric influenza hemagglutinin polypeptide comprising the stemdomain of A/Perth/16/2009 (H3).

5.10.3 Inactivated Virus Vaccines

In one embodiment, provided herein are immunogenic compositions (e.g.,vaccines) comprising an inactivated virus containing a chimericinfluenza hemagglutinin (HA) polypeptide described herein. In specificembodiments, the chimeric influenza hemagglutinin (HA) polypeptide ismembrane-bound. In particular embodiments, the inactivated virus is aninfluenza virus, such as described in Section 5.4, supra. In otherembodiments, the inactivated virus is a non-influenza virus, such asdescribed in Section 5.5, supra. In some embodiments, an immunogeniccomposition comprises two, three, four or more inactivated virusescontaining two, three, four or more different chimeric influenzahemagglutinin (HA) polypeptides described herein. In certainembodiments, the inactivated virus immunogenic compositions comprise oneor more adjuvants.

Techniques known to one of skill in the art may be used to inactivateviruses containing a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein. Common methods use formalin, heat, or detergent forinactivation. See, e.g., U.S. Pat. No. 6,635,246, which is hereinincorporated by reference in its entirety. Other methods include thosedescribed in U.S. Pat. Nos. 5,891,705; 5,106,619 and 4,693,981, whichare incorporated herein by reference in their entireties.

In certain embodiments, provided herein are immunogenic compositions(e.g., vaccines) comprising inactivated influenza virus such that eachdose of the immunogenic composition comprises about 7.5 μg to about 90μg, or about 15 to about 60 μg, of a chimeric influenza hemagglutinin(HA) polypeptide described herein, about 1.0 to about 5.0 mg sodiumchloride, about 20 to about 100 μg monobasic sodium phosphate, about 100to about 500 μg dibasic sodium phosphate, about 5 to about 30 μgmonobasic potassium phosphate, about 5 to about 30 μg potassiumchloride, and about 0.5 to about 3.0 μg calcium chloride. In someembodiments, the immunogenic compositions (e.g., vaccines) are packagedas single 0.25 ml or single 0.5 ml doses. In other embodiments, theimmunogenic compositions (e.g., vaccines) are packaged as multi-doseformulations.

In certain embodiments, provided herein are immunogenic compositions(e.g., vaccines) comprising inactivated influenza virus such that eachdose of the immunogenic composition comprises about 7.5 μg to about 90μg, or about 15 to about 60 μg, of a chimeric influenza hemagglutinin(HA) polypeptide described herein, about 0.001% to 0.01% thimerosal,about 1.0 to about 5.0 mg sodium chloride, about 20 to about 100 μgmonobasic sodium phosphate, about 100 to about 500 μg dibasic sodiumphosphate, about 5 to about 30 μg monobasic potassium phosphate, about 5to about 30 μg potassium chloride, and about 0.5 to about 3.0 μg calciumchloride per dose. In some embodiments, the immunogenic compositions(e.g., vaccines) are packaged as single 0.25 ml or single 0.5 ml doses.In other embodiments, the immunogenic compositions (e.g., vaccines) arepackaged as multi-dose formulations.

In a specific embodiment, immunogenic compositions (e.g., vaccines)provided herein are packaged as single 0.25 ml doses and comprise 22.5μg of a chimeric influenza hemagglutinin (HA) polypeptide describedherein, 2.05 mg sodium chloride, 40 μg monobasic sodium phosphate, 150μg dibasic sodium phosphate, 10 μg monobasic potassium phosphate, 10 μgpotassium chloride, and 0.75 μg calcium chloride per dose.

In a specific embodiment, immunogenic compositions (e.g., vaccines)provided herein are packaged as single 0.5 ml doses and comprise 45 μgof a chimeric influenza hemagglutinin (HA) polypeptide described herein,4.1 mg sodium chloride, 80 μg monobasic sodium phosphate, 300 μg dibasicsodium phosphate, 20 μg monobasic potassium phosphate, 20 μg potassiumchloride, and 1.5 μg calcium chloride per dose.

In a specific embodiment, immunogenic compositions (e.g., vaccines) arepackaged as multi-dose formulations comprising or consisting of 5.0 mlof vaccine (0.5 ml per dose) and comprise 24.5 μg of mercury (fromthimerosal), 45 μg of a chimeric influenza hemagglutinin (HA)polypeptide described herein, 4.1 mg sodium chloride, 80 μg monobasicsodium phosphate, 300 μg dibasic sodium phosphate, 20 μg monobasicpotassium phosphate, 20 μg potassium chloride, and 1.5 μg calciumchloride per dose.

In a specific embodiment, the inactivated virus that contains a chimericinfluenza hemagglutinin (HA) polypeptide described herein is propagatedin embryonated chicken eggs before its inactivation and subsequent usein an immunogenic composition described herein. In another specificembodiment, the inactivated virus that contains a chimeric influenzahemagglutinin (HA) polypeptide described herein is not propagated inembryonated chicken eggs before its inactivation and subsequent use inan immunogenic composition described herein. In another specificembodiment, the inactivated virus that contains a chimeric influenzahemagglutinin (HA) polypeptide described herein is propagated inmammalian cells, e.g., immortalized human cells (see, e.g.,International Application No. PCT/EP2006/067566 published asInternational Publication No. WO 07/045,674 which is herein incorporatedby reference in its entirety) or canine kidney cells such as MDCK cells(see, e.g., International Application No. PCT/IB2007/003536 published asInternational Publication No. WO 08/032,219 which is herein incorporatedby reference in its entirety) before its inactivation and subsequent usein an immunogenic composition described herein.

In a specific embodiment, provided herein is a monovalent inactivatedvirus vaccine comprising a chimeric influenza hemagglutinin (HA)polypeptide described herein. In a specific embodiment, provided hereinis a monovalent inactivated virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/1 chimericinfluenza hemagglutinin polypeptide (e.g., a cH5/1 chimeric influenzahemagglutinin polypeptide described in Section 5.1). In another specificembodiment, provided herein is a monovalent inactivated virus vaccinecomprising a virus that contains and/or comprises a genome that encodesa cH5/3 chimeric influenza hemagglutinin polypeptide (e.g., a cH5/3chimeric influenza hemagglutinin polypeptide described in Section 5.1).In another specific embodiment, provided herein is a monovalentinactivated virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH7/3 chimeric influenza hemagglutininpolypeptide (e.g., a cH7/3 chimeric influenza hemagglutinin polypeptidedescribed in Section 5.1). In another specific embodiment, providedherein is a monovalent inactivated virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/B chimericinfluenza hemagglutinin polypeptide (e.g., a cH5/B chimeric influenzahemagglutinin polypeptide described in Section 5.1). In another specificembodiment, provided herein is a monovalent inactivated virus vaccinecomprising a virus that contains and/or comprises a genome that encodesa cH7/B chimeric influenza hemagglutinin polypeptide (e.g., a cH7/1chimeric influenza hemagglutinin polypeptide described in Section 5.1).In another specific embodiment, provided herein is a monovalentinactivated virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cB/B chimeric influenza hemagglutininpolypeptide (e.g., a B/B chimeric influenza hemagglutinin polypeptidedescribed in Section 5.1).

In specific embodiments, provided herein are bivalent inactivated virusvaccines comprising viruses that contain and/or comprise a genome thatencodes a chimeric influenza hemagglutinin (HA) polypeptide describedherein. In a specific embodiment, provided herein is a bivalentinactivated virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH5/1 chimeric influenza hemagglutininpolypeptide and a virus that contains and/or comprises a genome thatencodes a cH5/3 chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a bivalent inactivated virusvaccine comprising a virus that contains and/or comprises a genome thatencodes a cH5/1 chimeric influenza hemagglutinin polypeptide and a virusthat contains and/or comprises a genome that encodes a cH7/3 chimericinfluenza hemagglutinin polypeptide. In another specific embodiment,provided herein is a bivalent inactivated virus vaccine comprising avirus that contains and/or comprises a genome that encodes a cH5/1chimeric influenza hemagglutinin polypeptide and a virus that containsand/or comprises a genome that encodes a cH5/B chimeric influenzahemagglutinin polypeptide. In another specific embodiment, providedherein is a bivalent inactivated virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a virus that containsand/or comprises a genome that encodes a cH5/1 chimeric influenzahemagglutinin polypeptide and a virus that contains and/or comprises agenome that encodes a cH7/B chimeric influenza hemagglutininpolypeptide. In another specific embodiment, provided herein is abivalent inactivated virus vaccine comprising a virus that containsand/or comprises a genome that encodes a cH5/1 chimeric influenzahemagglutinin polypeptide and a virus that contains and/or comprises agenome that encodes a cB/B chimeric influenza hemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalentinactivated virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH5/3 chimeric influenza hemagglutininpolypeptide and a virus that contains and/or comprises a genome thatencodes a cH7/3 chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a bivalent inactivated virusvaccine comprising a virus that contains and/or comprises a genome thatencodes a cH5/3 chimeric influenza hemagglutinin polypeptide and a virusthat contains and/or comprises a genome that encodes a cH5/B chimericinfluenza hemagglutinin polypeptide. In another specific embodiment,provided herein is a bivalent inactivated virus vaccine comprising avirus that contains and/or comprises a genome that encodes a cH5/3chimeric influenza hemagglutinin polypeptide and a virus that containsand/or comprises a genome that encodes a cH7/B chimeric influenzahemagglutinin polypeptide. In another specific embodiment, providedherein is a bivalent inactivated virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/3 chimericinfluenza hemagglutinin polypeptide and a virus that contains and/orcomprises a genome that encodes a cB/B chimeric influenza hemagglutininpolypeptide.

In another specific embodiment, provided herein is a bivalentinactivated virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH7/3 chimeric influenza hemagglutininpolypeptide and a virus that contains and/or comprises a genome thatencodes a cH5/B chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a bivalent inactivated virusvaccine comprising a virus that contains and/or comprises a genome thatencodes a cH7/3 chimeric influenza hemagglutinin polypeptide and a virusthat contains and/or comprises a genome that encodes a cH7/B chimericinfluenza hemagglutinin polypeptide. In another specific embodiment,provided herein is a bivalent inactivated virus vaccine comprising avirus that contains and/or comprises a genome that encodes a cH7/3chimeric influenza hemagglutinin polypeptide and a virus that containsand/or comprises a genome that encodes a cB/B chimeric influenzahemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalentinactivated virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH5/B chimeric influenza hemagglutininpolypeptide and a virus that contains and/or comprises a genome thatencodes a cH7/B chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a bivalent inactivated virusvaccine comprising a virus that contains and/or comprises a genome thatencodes a cH5/B chimeric influenza hemagglutinin polypeptide and a virusthat contains and/or comprises a genome that encodes a cB/B chimericinfluenza hemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalentinactivated virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH7/B chimeric influenza hemagglutininpolypeptide and a virus that contains and/or comprises a genome thatencodes a cB/B chimeric influenza hemagglutinin polypeptide.

In specific embodiments, provided herein are trivalent inactivated virusvaccines comprising viruses that contain and/or comprise a genome thatencodes a chimeric influenza hemagglutinin (HA) polypeptide describedherein. In a specific embodiment, provided herein is a trivalentinactivated virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH5/1 chimeric influenza hemagglutininpolypeptide, a virus that contains and/or comprises a genome thatencodes a cH5/3 chimeric influenza hemagglutinin polypeptide, and avirus that contains and/or comprises a genome that encodes a cH5/Bchimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a trivalent inactivated virus vaccinecomprising a virus that contains and/or comprises a genome that encodesa cH5/1 chimeric influenza hemagglutinin polypeptide, a virus thatcontains and/or comprises a genome that encodes a cH5/3 chimericinfluenza hemagglutinin polypeptide, and a virus that contains and/orcomprises a genome that encodes a cH7/B chimeric influenza hemagglutininpolypeptide. In another specific embodiment, provided herein is atrivalent inactivated virus vaccine comprising a virus that containsand/or comprises a genome that encodes a cH5/1 chimeric influenzahemagglutinin polypeptide, a virus that contains and/or comprises agenome that encodes a cH5/3 chimeric influenza hemagglutininpolypeptide, and a virus that contains and/or comprises a genome thatencodes a cB/B chimeric influenza hemagglutinin polypeptide.

In another specific embodiment, provided herein is a trivalentinactivated virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH5/1 chimeric influenza hemagglutininpolypeptide, a virus that contains and/or comprises a genome thatencodes a cH7/3 chimeric influenza hemagglutinin polypeptide, and avirus that contains and/or comprises a genome that encodes a cH5/Bchimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a trivalent inactivated virus vaccinecomprising a virus that contains and/or comprises a genome that encodesa cH5/1 chimeric influenza hemagglutinin polypeptide, a virus thatcontains and/or comprises a genome that encodes a cH7/3 chimericinfluenza hemagglutinin polypeptide, and a virus that contains and/orcomprises a genome that encodes a cH7/B chimeric influenza hemagglutininpolypeptide. In another specific embodiment, provided herein is atrivalent inactivated virus vaccine comprising a virus that containsand/or comprises a genome that encodes a cH5/1 chimeric influenzahemagglutinin polypeptide, a virus that contains and/or comprises agenome that encodes a cH7/3 chimeric influenza hemagglutininpolypeptide, and a virus that contains and/or comprises a genome thatencodes a cB/B chimeric influenza hemagglutinin polypeptide.

In a specific embodiment, an inactivated virus vaccine provided hereindoes not comprise a virus that contains and/or comprises a genome thatencodes a cH5/3 chimeric influenza hemagglutinin polypeptide comprisingthe globular head domain of A/Vietnam/1203/2004 (H5). In anotherspecific embodiment, an inactivated virus vaccine provided herein doesnot comprise a virus that contains and/or comprises a genome thatencodes a cH5/3 chimeric influenza hemagglutinin polypeptide comprisingthe stem domain of A/Perth/16/2009 (H3).

In a specific embodiment, an inactivated virus vaccine provided hereindoes not comprise a virus that contains and/or comprises a genome thatencodes a cH7/3 chimeric influenza hemagglutinin polypeptide comprisingthe globular head domain of A/mallard/Alberta/24/2001 (H7). In anotherspecific embodiment, an inactivated virus vaccine provided herein doesnot comprise a virus that contains and/or comprises a genome thatencodes a cH7/3 chimeric influenza hemagglutinin polypeptide comprisingthe stem domain of A/Perth/16/2009 (H3).

5.10.4 Split Virus Vaccines

In one embodiment, an immunogenic composition comprising a chimericinfluenza hemagglutinin (HA) polypeptide described herein is a splitvirus vaccine. In some embodiments, split virus vaccine contains two,three, four or more different chimeric influenza hemagglutinin (HA)polypeptides described herein. In certain embodiments, the chimericinfluenza hemagglutinin (HA) polypeptide is/was membrane-bound. Incertain embodiments, the split virus vaccines comprise one or moreadjuvants.

Techniques for producing split virus vaccines are known to those skilledin the art. By way of non-limiting example, an influenza virus splitvaccine may be prepared using inactivated particles disrupted withdetergents. One example of a split virus vaccine that can be adapted foruse in accordance with the methods described herein is the Fluzone®,Influenza Virus Vaccine (Zonal Purified, Subvirion) for intramuscularuse, which is formulated as a sterile suspension prepared from influenzaviruses propagated in embryonated chicken eggs. The virus-containingfluids are harvested and inactivated with formaldehyde. Influenza virusis concentrated and purified in a linear sucrose density gradientsolution using a continuous flow centrifuge. The virus is thenchemically disrupted using a nonionic surfactant, octoxinol-9, (Triton®X-100—A registered trademark of Union Carbide, Co.) producing a “splitvirus.” The split virus is then further purified by chemical means andsuspended in sodium phosphate-buffered isotonic sodium chloridesolution.

In certain embodiments, provided herein are split virus vaccinescomprising about 7.5 μg to about 90 μg, or 10 μg to about 60 μg, of oneor more chimeric influenza hemagglutinin (HA) polypeptide describedherein, about 0.01 to about 1.0 mg octoxynol-10 (TRITON X-100®, about0.5 to 0.5 mg α-tocopheryl hydrogen succinate, about 0.1 to 1.0 mgpolysorbate 80 (Tween 80), about 0.001 to about 0.003 μg hydrocortisone,about 0.05 to about 0.3 μg gentamcin sulfate, about 0.5 to about 2.0 μgchicken egg protein (ovalbumin), about 25 to 75 μg formaldehyde, andabout 25 to 75 μg sodium deoxycholate.

In a specific embodiment, a split virus vaccine provided hereincomprises or consists of a 0.5 ml dose that comprises 45 μg of achimeric influenza hemagglutinin (HA) polypeptide described herein,≦0.085 mg octoxynol-10 (TRITON X-100®, ≦0.1 mg α-tocopheryl hydrogensuccinate, ≦0.415 mg polysorbate 80 (Tween 80), ≦0.0016 μghydrocortisone, ≦0.15 μg gentamcin sulfate, ≦1.0 chicken egg protein(ovalbumin), ≦50 μg formaldehyde, and ≦50 μg sodium deoxycholate. Insome embodiments, the 0.5 ml dose subunit vaccine is packaged in apre-filled syringe.

In a specific embodiment, the split virus vaccine is prepared usinginfluenza virus that was propagated in embryonated chicken eggs. Inanother specific embodiment, the split virus vaccine is prepared usinginfluenza virus that was not propagated in embryonated chicken eggs. Inanother specific embodiment, the split virus vaccine is prepared usinginfluenza virus that was propagated in mammalian cells, e.g.,immortalized human cells (see, e.g., PCT/EP2006/067566 published as WO07/045,674 which is herein incorporated by reference in its entirety) orcanine kidney cells such as MDCK cells (see, e.g., PCT/IB2007/003536published as WO 08/032,219 which is herein incorporated by reference inits entirety).

In a specific embodiment, provided herein is a monovalent split virusvaccine comprising a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein. In a specific embodiment, provided herein is amonovalent split virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH5/1 chimeric influenza hemagglutininpolypeptide (e.g., a cH5/1 chimeric influenza hemagglutinin polypeptidedescribed in Section 5.1). In another specific embodiment, providedherein is a monovalent split virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/3 chimericinfluenza hemagglutinin polypeptide (e.g., a cH5/3 chimeric influenzahemagglutinin polypeptide described in Section 5.1). In another specificembodiment, provided herein is a monovalent split virus vaccinecomprising a virus that contains and/or comprises a genome that encodesa cH7/3 chimeric influenza hemagglutinin polypeptide (e.g., a cH7/3chimeric influenza hemagglutinin polypeptide described in Section 5.1).In another specific embodiment, provided herein is a monovalent splitvirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH5/B chimeric influenza hemagglutinin polypeptide (e.g.,a cH5/B chimeric influenza hemagglutinin polypeptide described inSection 5.1). In another specific embodiment, provided herein is amonovalent split virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH7/B chimeric influenza hemagglutininpolypeptide (e.g., a cH7/1 chimeric influenza hemagglutinin polypeptidedescribed in Section 5.1). In another specific embodiment, providedherein is a monovalent split virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cB/B chimericinfluenza hemagglutinin polypeptide (e.g., a B/B chimeric influenzahemagglutinin polypeptide described in Section 5.1).

In specific embodiments, provided herein are bivalent split virusvaccines comprising viruses that contain and/or comprise a genome thatencodes a chimeric influenza hemagglutinin (HA) polypeptide describedherein. In a specific embodiment, provided herein is a bivalent splitvirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH5/1 chimeric influenza hemagglutinin polypeptide and avirus that contains and/or comprises a genome that encodes a cH5/3chimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a bivalent split virus vaccine comprisinga virus that contains and/or comprises a genome that encodes a cH5/1chimeric influenza hemagglutinin polypeptide and a virus that containsand/or comprises a genome that encodes a cH7/3 chimeric influenzahemagglutinin polypeptide. In another specific embodiment, providedherein is a bivalent split virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/1 chimericinfluenza hemagglutinin polypeptide and a virus that contains and/orcomprises a genome that encodes a cH5/B chimeric influenza hemagglutininpolypeptide. In another specific embodiment, provided herein is abivalent split virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a virus that contains and/or comprises agenome that encodes a cH5/1 chimeric influenza hemagglutinin polypeptideand a virus that contains and/or comprises a genome that encodes a cH7/Bchimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a bivalent split virus vaccine comprisinga virus that contains and/or comprises a genome that encodes a cH5/1chimeric influenza hemagglutinin polypeptide and a virus that containsand/or comprises a genome that encodes a cB/B chimeric influenzahemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalent splitvirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH5/3 chimeric influenza hemagglutinin polypeptide and avirus that contains and/or comprises a genome that encodes a cH7/3chimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a bivalent split virus vaccine comprisinga virus that contains and/or comprises a genome that encodes a cH5/3chimeric influenza hemagglutinin polypeptide and a virus that containsand/or comprises a genome that encodes a cH5/B chimeric influenzahemagglutinin polypeptide. In another specific embodiment, providedherein is a bivalent split virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/3 chimericinfluenza hemagglutinin polypeptide and a virus that contains and/orcomprises a genome that encodes a cH7/B chimeric influenza hemagglutininpolypeptide. In another specific embodiment, provided herein is abivalent split virus vaccine comprising a virus that contains and/orcomprises a genome that encodes a cH5/3 chimeric influenza hemagglutininpolypeptide and a virus that contains and/or comprises a genome thatencodes a cB/B chimeric influenza hemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalent splitvirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH7/3 chimeric influenza hemagglutinin polypeptide and avirus that contains and/or comprises a genome that encodes a cH5/Bchimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a bivalent split virus vaccine comprisinga virus that contains and/or comprises a genome that encodes a cH7/3chimeric influenza hemagglutinin polypeptide and a virus that containsand/or comprises a genome that encodes a cH7/B chimeric influenzahemagglutinin polypeptide. In another specific embodiment, providedherein is a bivalent split virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH7/3 chimericinfluenza hemagglutinin polypeptide and a virus that contains and/orcomprises a genome that encodes a cB/B chimeric influenza hemagglutininpolypeptide.

In another specific embodiment, provided herein is a bivalent splitvirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH5/B chimeric influenza hemagglutinin polypeptide and avirus that contains and/or comprises a genome that encodes a cH7/Bchimeric influenza hemagglutinin polypeptide. In another specificembodiment, provided herein is a bivalent split virus vaccine comprisinga virus that contains and/or comprises a genome that encodes a cH5/Bchimeric influenza hemagglutinin polypeptide and a virus that containsand/or comprises a genome that encodes a cB/B chimeric influenzahemagglutinin polypeptide.

In another specific embodiment, provided herein is a bivalent splitvirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH7/B chimeric influenza hemagglutinin polypeptide and avirus that contains and/or comprises a genome that encodes a cB/Bchimeric influenza hemagglutinin polypeptide.

In specific embodiments, provided herein are trivalent split virusvaccines comprising viruses that contain and/or comprise a genome thatencodes a chimeric influenza hemagglutinin (HA) polypeptide describedherein. In a specific embodiment, provided herein is a trivalent splitvirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH5/1 chimeric influenza hemagglutinin polypeptide, avirus that contains and/or comprises a genome that encodes a cH5/3chimeric influenza hemagglutinin polypeptide, and a virus that containsand/or comprises a genome that encodes a cH5/B chimeric influenzahemagglutinin polypeptide. In another specific embodiment, providedherein is a trivalent split virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/1 chimericinfluenza hemagglutinin polypeptide, a virus that contains and/orcomprises a genome that encodes a cH5/3 chimeric influenza hemagglutininpolypeptide, and a virus that contains and/or comprises a genome thatencodes a cH7/B chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a trivalent split virus vaccinecomprising a virus that contains and/or comprises a genome that encodesa cH5/1 chimeric influenza hemagglutinin polypeptide, a virus thatcontains and/or comprises a genome that encodes a cH5/3 chimericinfluenza hemagglutinin polypeptide, and a virus that contains and/orcomprises a genome that encodes a cB/B chimeric influenza hemagglutininpolypeptide.

In another specific embodiment, provided herein is a trivalent splitvirus vaccine comprising a virus that contains and/or comprises a genomethat encodes a cH5/1 chimeric influenza hemagglutinin polypeptide, avirus that contains and/or comprises a genome that encodes a cH7/3chimeric influenza hemagglutinin polypeptide, and a virus that containsand/or comprises a genome that encodes a cH5/B chimeric influenzahemagglutinin polypeptide. In another specific embodiment, providedherein is a trivalent split virus vaccine comprising a virus thatcontains and/or comprises a genome that encodes a cH5/1 chimericinfluenza hemagglutinin polypeptide, a virus that contains and/orcomprises a genome that encodes a cH7/3 chimeric influenza hemagglutininpolypeptide, and a virus that contains and/or comprises a genome thatencodes a cH7/B chimeric influenza hemagglutinin polypeptide. In anotherspecific embodiment, provided herein is a trivalent split virus vaccinecomprising a virus that contains and/or comprises a genome that encodesa cH5/1 chimeric influenza hemagglutinin polypeptide, a virus thatcontains and/or comprises a genome that encodes a cH7/3 chimericinfluenza hemagglutinin polypeptide, and a virus that contains and/orcomprises a genome that encodes a cB/B chimeric influenza hemagglutininpolypeptide.

In a specific embodiment, a split virus vaccine provided herein does notcomprise a virus that contains and/or comprises a genome that encodes acH5/3 chimeric influenza hemagglutinin polypeptide comprising theglobular head domain of A/Vietnam/1203/2004 (H5). In another specificembodiment, a split virus vaccine provided herein does not comprise avirus that contains and/or comprises a genome that encodes a cH5/3chimeric influenza hemagglutinin polypeptide comprising the stem domainof A/Perth/16/2009 (H3).

In a specific embodiment, a split virus vaccine provided herein does notcomprise a virus that contains and/or comprises a genome that encodes acH7/3 chimeric influenza hemagglutinin polypeptide comprising theglobular head domain of A/mallard/Alberta/24/2001 (H7). In anotherspecific embodiment, a split virus vaccine provided herein does notcomprise a virus that contains and/or comprises a genome that encodes acH7/3 chimeric influenza hemagglutinin polypeptide comprising the stemdomain of A/Perth/16/2009 (H3).

5.10.5 Adjuvants

In certain embodiments, the compositions described herein comprise, orare administered in combination with, an adjuvant. The adjuvant foradministration in combination with a composition described herein may beadministered before, concommitantly with, or after administration ofsaid composition. In some embodiments, the term “adjuvant” refers to acompound that when administered in conjunction with or as part of acomposition described herein augments, enhances and/or boosts the immuneresponse to a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein, but when the compound is administered alone does notgenerate an immune response to the polypeptide. In some embodiments, theadjuvant generates an immune response to the polypeptide and does notproduce an allergy or other adverse reaction. Adjuvants can enhance animmune response by several mechanisms including, e.g., lymphocyterecruitment, stimulation of B and/or T cells, and stimulation ofmacrophages.

In certain embodiments, an adjuvant augments the intrinsic response tochimeric influenza hemagglutinin (HA) polypeptide without causingconformational changes in the polypeptide that affect the qualitativeform of the response. Specific examples of adjuvants include, but arenot limited to, aluminum salts (alum) (such as aluminum hydroxide,aluminum phosphate, and aluminum sulfate), 3 De-O-acylatedmonophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03(GlaxoSmithKline), A504 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICLAmericas, Inc.), imidazopyridine compounds (see InternationalApplication No. PCT/US2007/064857, published as InternationalPublication No. WO2007/109812), imidazoquinoxaline compounds (seeInternational Application No. PCT/US2007/064858, published asInternational Publication No. WO2007/109813) and saponins, such as QS21(see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach(eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No.5,057,540). In some embodiments, the adjuvant is Freund's adjuvant(complete or incomplete). Other adjuvants are oil in water emulsions(such as squalene or peanut oil), optionally in combination with immunestimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl.J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (Bioworld Today,Nov. 15, 1998). Such adjuvants can be used with or without otherspecific immunostimulating agents such as MPL or 3-DMP, QS21, polymericor monomeric amino acids such as polyglutamic acid or polylysine, orother immunopotentiating agents described in Section 5.4, supra. Itshould be understood that different formulations of chimeric influenzahemagglutinin (HA) polypeptides described herein may comprise differentadjuvants or may comprise the same adjuvant.

5.11 Prophylactic and Therapeutic Uses

In one aspect, provided herein are methods for inducing an immuneresponse in a subject utilizing an active compound (e.g., a chimericinfluenza hemagglutinin (HA) polypeptide described herein, a nucleicacid encoding such a polypeptide, a vector (e.g., a viral vector, or abacteria) containing or expressing such a polypeptide, cells stimulatedwith such a polypeptide) or a composition (e.g., vaccine formulation)described herein. In a specific embodiment, a method for inducing animmune response to an influenza virus hemagglutinin (HA) polypeptide ina subject comprises administering to a subject in need thereof aneffective amount of a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein or an immunogenic composition thereof (e.g., a vaccineformulation thereof). In another embodiment, a method for inducing animmune response to an influenza virus hemagglutinin polypeptide in asubject comprises administering to a subject in need thereof aneffective amount of a nucleic acid encoding a chimeric influenzahemagglutinin (HA) polypeptide described herein or an immunogeniccomposition thereof. In another embodiment, a method for inducing animmune response to an influenza virus hemagglutinin polypeptide in asubject comprises administering to a subject in need thereof aneffective amount of a viral vector containing or expressing a chimericinfluenza hemagglutinin (HA) polypeptide described herein or animmunogenic composition thereof. In yet another embodiment, a method forinducing an immune response to an influenza virus hemagglutininpolypeptide in a subject comprises administering to a subject in needthereof an effective amount of cells stimulated with a chimericinfluenza hemagglutinin (HA) polypeptide described herein or apharmaceutical composition thereof. In certain embodiments, a chimericinfluenza hemagglutinin (HA) polypeptide described herein used in themethod is a purified chimeric influenza hemagglutinin (HA) polypeptidedescribed herein derived from a mammalian cell, a plant cell, or aninsect cell.

In a specific embodiment, a method for inducing an immune response to aninfluenza virus hemagglutinin polypeptide in a subject comprisesadministering to a subject in need thereof a subunit vaccine describedherein (see Section 5.10.1). In another embodiment, a method forinducing an immune response to an influenza virus hemagglutininpolypeptide in a subject comprises administering to a subject in needthereof a live virus vaccine described herein (see Section 5.10.2). Inparticular embodiments, the live virus vaccine comprises an attenuatedvirus. In another embodiment, a method for inducing an immune responseto an influenza virus hemagglutinin polypeptide in a subject comprisesadministering to a subject in need thereof an inactivated virus vaccinedescribed herein (see Section 5.10.3). In another embodiment, a methodfor inducing an immune response to an influenza virus hemagglutininpolypeptide in a subject comprises administering to a subject in needthereof a split virus vaccine described herein (see Section 5.10.4). Inanother embodiment, a method for inducing an immune response to aninfluenza virus hemagglutinin polypeptide in a subject comprisesadministering to a subject in need thereof a virus-like particle vaccinedescribed herein (see Section 5.6). In another embodiment, a method forinducing an immune response to an influenza hemagglutinin polypeptidecomprises administering to a subject in need thereof a virosomedescribed herein (see Section 5.6). In another embodiment, a method forinducing an immune response to an influenza hemagglutinin polypeptidecomprises administering to a subject in need thereof a bacteriaexpressing or engineered to express a chimeric influenza hemagglutinin(HA) polypeptide described herein or a composition thereof (see Section5.7). In certain embodiments, a chimeric influenza hemagglutinin (HA)polypeptide described herein used in the method is a purified chimericinfluenza hemagglutinin (HA) polypeptide described herein derived from amammalian cell, a plant cell, or an insect cell.

In some embodiments, the immune response induced by an active compound(e.g, a chimeric influenza hemagglutinin (HA) polypeptide describedherein, a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or a composition(e.g., vaccine formulation) described herein is effective to preventand/or treat an influenza virus infection caused by any subtype orstrain of influenza virus. In some embodiments, the immune responseinduced by an active compound or a composition (e.g., vaccineformulation) described herein is effective to prevent and/or treat aninfluenza virus infection caused by one or more strains within the samesubtype of influenza virus. In some embodiments, the immune responseinduced by an active compound or a composition (e.g., vaccineformulation) described herein is effective to prevent and/or treat aninfluenza virus infection caused by multiple strains of the same subtypeof influenza virus.

In some embodiments, the immune response induced by an active compound(e.g, a chimeric influenza hemagglutinin (HA) polypeptide describedherein, a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or a composition(e.g., vaccine formulation) described herein is effective to preventand/or treat an influenza virus infection caused by (i) H1N1 and H1N2subtypes; (ii) H1N1 and H3N1 subtypes; (iii) H1N2 and H3N2 subtypes;and/or (iv) any of the combinations of (i)-(iii) and influenza B.

In certain embodiments, the immune response induced by an activecompound or a composition (e.g., vaccine formulation) described hereinis effective to prevent and/or treat an influenza virus infection causedby a subtype of influenza virus that belongs to one HA group (e.g.,Group 1, which comprises H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16)and not the other HA group (e.g., Group 2, which comprises H3, H4, H7,H10, H14, and H15). For example, the immune response induced may beeffective to prevent and/or treat an influenza virus infection caused byan influenza virus that belongs to the HA group consisting of H11, H13,H16, H9, H8, H12, H6, H1, H5 and/or H2. Alternatively, the immuneresponse induced may be effective to prevent and/or treat an influenzavirus infection caused by an influenza virus that belongs to the HAgroup consisting of H3, H4, H14, H10, H15 and/or H7. In someembodiments, the immune response induced by an active compound or acomposition (e.g., vaccine formulation) described herein is effective toprevent and/or treat an influenza virus infection caused by one, two,three, four or five subtypes of influenza virus. In certain embodiments,the immune response induced by an active compound or a composition(e.g., vaccine formulation) described herein is effective to preventand/or treat an influenza virus infection caused by six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen or fifteen subtypes ofinfluenza virus. In a specific embodiment, the immune response inducedby an active compound or a composition (e.g., vaccine formulation)described herein is effective to prevent and/or treat an influenza virusinfection caused by one, two, or more strains of an influenza virus thatbelongs to the H1 HA group and/or one, two, or more strains of aninfluenza virus that belongs to the H2 HA group. In another specificembodiment, the immune response induced by an active compound or acomposition (e.g., vaccine formulation) described herein is effective toprevent and/or treat an influenza virus infection caused by one, two, ormore strains of an influenza virus that belongs to the H1 HA group; one,two, or more strains of an influenza virus that belongs to the H3 HAgroup; and/or one, two, or more influenza B virus strains.

In some embodiments, the immune response induced by an active compound(e.g, a chimeric influenza hemagglutinin (HA) polypeptide describedherein, a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or a composition(e.g., vaccine formulation) described herein is effective to preventand/or treat an influenza virus infection caused by both H1N1 and H2N2subtypes. In other embodiments, the immune response induced by an activecompound or a composition (e.g., vaccine formulation) described hereinis not effective to prevent and/or treat an influenza virus infectioncaused by both H1N1 and H2N2 subtypes. In some embodiments, the immuneresponse induced by an active compound or a composition (e.g., vaccineformulation) described herein is effective to prevent and/or treat aninfluenza virus infection caused by H1N1, H2N2, and H3N2 subtypes. Insome embodiments, the immune response induced by an active compound or acomposition (e.g., vaccine formulation) described herein is effective toprevent and/or treat an influenza virus infection caused by H3N2subtypes. In other embodiments, the immune response induced by an activecompound or a composition (e.g., vaccine formulation) described hereinis not effective to prevent and/or treat an influenza virus infectioncaused by H3N2 subtypes.

In certain embodiments, the immune response induced by an activecompound or a composition (e.g., vaccine formulation) described hereinis effective to prevent and/or treat an influenza virus disease causedby a subtype of influenza virus that belongs to one HA group and not theother HA group. For example, the immune response induced may beeffective to prevent and/or treat an influenza virus disease caused byan influenza virus that belongs to the HA group consisting of H11, H13,H16, H9, H8, H12, H6, H1, H5 and/or H2. Alternatively, the immuneresponse induced may be effective to prevent and/or treat an influenzavirus disease caused by an influenza virus that belongs to the HA groupconsisting of H3, H4, H14, H10, H15 and/or H7. In some embodiments, theimmune response induced by an active compound or a composition (e.g.,vaccine formulation) described herein is effective to prevent and/ortreat an influenza virus disease caused by any of one, two, three, fouror five subtypes of influenza virus. In certain embodiments, the immuneresponse induced by an active compound or a composition (e.g., vaccineformulation) described herein is effective to prevent and/or treat aninfluenza virus disease caused by any of six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen or fifteen subtypes of influenzavirus. In some embodiments, the immune response induced by an activecompound or a composition (e.g., vaccine formulation) described hereinis effective to prevent and/or treat an influenza virus disease causedby one or more strains within the same subtype of influenza virus.

In some embodiments, the immune response induced by an active compound(e.g, a chimeric influenza hemagglutinin (HA) polypeptide describedherein, a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or a composition(e.g., vaccine formulation) described herein is effective to reducesymptoms resulting from an influenza virus disease/infection. Symptomsof influenza virus disease/infection include, but are not limited to,body aches (especially joints and throat), fever, nausea, headaches,irritated eyes, fatigue, sore throat, reddened eyes or skin, andabdominal pain.

In some embodiments, the immune response induced by an active compound(e.g, a chimeric influenza hemagglutinin (HA) polypeptide describedherein, a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or a composition(e.g., vaccine formulation) described herein is effective to reduce thehospitalization of a subject suffering from an influenza virusdisease/infection. In some embodiments, the immune response induced byan active compound or a composition (e.g., vaccine formulation)described herein is effective to reduce the duration of hospitalizationof a subject suffering from an influenza virus disease/infection.

In another aspect, provided herein are methods for preventing and/ortreating an influenza virus infection in a subject utilizing an activecompound (e.g, a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein, a nucleic acid encoding such a polypeptide, a vector(e.g., a viral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or a composition(e.g., vaccine formulation) described herein. In one embodiment, amethod for preventing or treating an influenza virus infection in asubject comprises administering to a subject in need thereof a chimericinfluenza hemagglutinin (HA) polypeptide described herein, a nucleicacid encoding such a polypeptide, a vector containing or expressing sucha polypeptide, or a composition of any one of the foregoing. In aspecific embodiment, a method for preventing or treating an influenzavirus infection in a subject comprises administering to a subject inneed thereof a subunit vaccine, a live virus vaccine, an inactivatedvirus vaccine, a split virus vaccine or a virus-like particle vaccinedescribed herein.

In another aspect, provided herein are methods for preventing and/ortreating an influenza virus disease in a subject utilizing a chimericinfluenza hemagglutinin (HA) polypeptide described herein, a nucleicacid encoding such a polypeptide, a vector containing or expressing sucha polypeptide, or cells stimulated with such a polypeptide, or acomposition (e.g., vaccine formulation) described herein. In a specificembodiment, a method for preventing or treating an influenza virusdisease in a subject comprises administering to a subject in needthereof an effective amount of a chimeric influenza hemagglutinin (HA)polypeptide described herein or an immunogenic composition thereof. Inanother embodiment, a method for preventing or treating an influenzavirus disease in a subject comprises administering to a subject in needthereof an effective amount of a nucleic acid encoding a chimericinfluenza hemagglutinin (HA) polypeptide described herein or animmunogenic composition thereof. In another embodiment, a method forpreventing or treating an influenza virus disease in a subject comprisesadministering to a subject in need thereof an effective amount of aviral vector containing or expressing a chimeric influenza hemagglutinin(HA) polypeptide described herein or an immunogenic composition thereof.In yet another embodiment, a method for preventing or treating aninfluenza virus disease in a subject comprises administering to asubject in need thereof an effective amount of cells stimulated with achimeric influenza hemagglutinin (HA) polypeptide described herein or apharmaceutical composition thereof.

In a specific embodiment, a method for preventing or treating aninfluenza virus disease in a subject comprises administering to asubject in need thereof a subunit vaccine described herein. In anotherembodiment, a method for preventing or treating an influenza virusdisease in a subject comprises administering to a subject in needthereof a live virus vaccine described herein. In particularembodiments, the live virus vaccine comprises an attenuated virus. Inanother embodiment, a method for preventing or treating an influenzavirus disease in a subject comprises administering to a subject in needthereof an inactivated virus vaccine described herein. In anotherembodiment, a method for preventing or treating an influenza virusdisease in a subject comprises administering to a subject in needthereof a split virus vaccine described herein. In another embodiment, amethod for preventing or treating an influenza virus disease comprisesadministering to a subject in need thereof a virus-like particle vaccinedescribed herein. In another embodiment, a method for preventing ortreating an influenza virus disease in a subject, comprisingadministering to a subject in need thereof a virosome described herein.In another embodiment, a method for preventing or treating an influenzavirus disease in a subject comprising administering to a subject in needthereof a bacteria expressing or engineered to express a chimericinfluenza hemagglutinin (HA) polypeptide described herein or acomposition thereof.

In another aspect, provided herein are methods of preventing and/ortreating an influenza virus disease in a subject by administeringneutralizing antibodies described herein (see Section 5.9). In aspecific embodiment, a method for preventing or treating an influenzavirus disease in a subject comprises administering to a subject in needthereof an effective amount of a neutralizing antibody described herein,or a pharmaceutical composition thereof. In particular embodiments, theneutralizing antibody is a monoclonal antibody. In certain embodiments,the neutralizing antibody is not CR6261, CR6325, CR6329, CR6307, CR6323,2A, D7, D8, F10, G17, H40, A66, D80, E88, E90, H98, C179 (FERM BP-4517),AI3C (FERM BP-4516) or any other antibody described in Ekiert D C et al.(2009) Antibody Recognition of a Highly Conserved Influenza VirusEpitope. Science (published in Science Express Feb. 26, 2009); Kashyapet al. (2008) Combinatorial antibody libraries from survivors of theTurkish H5N1 avian influenza outbreak reveal virus neutralizationstrategies. Proc Natl Acad Sci USA 105: 5986-5991; Sui et al. (2009)Structural and functional bases for broad-spectrum neutralization ofavian and human influenza A viruses. Nat Struct Mol Biol 16: 265-273;U.S. Pat. Nos. 5,589,174, 5,631,350, 6,337,070, and 6,720,409;International Application No. PCT/US2007/068983 published asInternational Publication No. WO 2007/134237; International ApplicationNo. PCT/US2008/075998 published as International Publication No. WO2009/036157; International Application No. PCT/EP2007/059356 publishedas International Publication No. WO 2008/028946; and InternationalApplication No. PCT/US2008/085876 published as International PublicationNo. WO 2009/079259. In other embodiments, the neutralizing antibody isnot an antibody described in Wang et al. (2010) “Broadly ProtectiveMonoclonal Antibodies against H3 Influenza Viruses following SequentialImmunization with Different Hemagglutinins,” PLOS Pathogens 6(2):1-9.

In certain embodiments, the methods for preventing or treating aninfluenza virus disease or infection in a subject (e.g., a human ornon-human animal) provided herein result in a reduction in thereplication of the influenza virus in the subject as measured by in vivoand in vitro assays known to those of skill in the art and describedherein. In some embodiments, the replication of the influenza virus isreduced by approximately 1 log or more, approximately 2 logs or more,approximately 3 logs or more, approximately 4 logs or more,approximately 5 logs or more, approximately 6 logs or more,approximately 7 logs or more, approximately 8 logs or more,approximately 9 logs or more, approximately 10 logs or more, 1 to 3logs, 1 to 5 logs, 1 to 8 logs, 1 to 9 logs, 2 to 10 logs, 2 to 5 logs,2 to 7 logs, 2 logs to 8 logs, 2 to 9 logs, 2 to 10 logs 3 to 5 logs, 3to 7 logs, 3 to 8 logs, 3 to 9 logs, 4 to 6 logs, 4 to 8 logs, 4 to 9logs, 5 to 6 logs, 5 to 7 logs, 5 to 8 logs, 5 to 9 logs, 6 to 7 logs, 6to 8 logs, 6 to 9 logs, 7 to 8 logs, 7 to 9 logs, or 8 to 9 logs.

5.11.1 Combination Therapies

In various embodiments, a chimeric influenza hemagglutinin (HA)polypeptide described herein, a nucleic acid encoding such apolypeptide, a vector (e.g., a viral vector or a bacteria) containing orexpressing such a polypeptide, cells stimulated with such a polypeptide,or a neutralizing antibody may be administered to a subject incombination with one or more other therapies (e.g., antiviral,antibacterial, or immunomodulatory therapies). In some embodiments, apharmaceutical composition (e.g., an immunogenic composition) describedherein may be administered to a subject in combination with one or moretherapies. The one or more other therapies may be beneficial in thetreatment or prevention of an influenza virus disease or may amelioratea symptom or condition associated with an influenza virus disease. Insome embodiments, the one or more other therapies are pain relievers,anti-fever medications, or therapies that alleviate or assist withbreathing. In certain embodiments, the therapies are administered lessthan 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about1 hour apart, at about 1 to about 2 hours apart, at about 2 hours toabout 3 hours apart, at about 3 hours to about 4 hours apart, at about 4hours to about 5 hours apart, at about 5 hours to about 6 hours apart,at about 6 hours to about 7 hours apart, at about 7 hours to about 8hours apart, at about 8 hours to about 9 hours apart, at about 9 hoursto about 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours part. In specificembodiments, two or more therapies are administered within the samepatent visit.

Any anti-viral agents well-known to one of skill in the art may used incombination with an active compound (e.g, a chimeric influenzahemagglutinin (HA) polypeptide described herein, a nucleic acid encodingsuch a polypeptide, a vector (e.g., a viral vector, or a bacteria)containing or expressing such a polypeptide, cells stimulated with sucha polypeptide) or pharmaceutical composition described herein.Non-limiting examples of anti-viral agents include proteins,polypeptides, peptides, fusion proteins antibodies, nucleic acidmolecules, organic molecules, inorganic molecules, and small moleculesthat inhibit and/or reduce the attachment of a virus to its receptor,the internalization of a virus into a cell, the replication of a virus,or release of virus from a cell. In particular, anti-viral agentsinclude, but are not limited to, nucleoside analogs (e.g., zidovudine,acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, andribavirin), foscarnet, amantadine, peramivir, rimantadine, saquinavir,indinavir, ritonavir, alpha-interferons and other interferons, AZT,zanamivir (Relenza®), and oseltamivir (Tamiflu®). Other anti-viralagents include influenza virus vaccines, e.g., Fluarix®(GlaxoSmithKline), FluMist® (MedImmune Vaccines), Fluvirin® (ChironCorporation), Flulaval® (GlaxoSmithKline), Afluria® (CSL BiotherapiesInc.), Agriflu® (Novartis) or Fluzone® (Aventis Pasteur).

In specific embodiments, the anti-viral agent is an immunomodulatoryagent that is specific for a viral antigen. In particular embodiments,the viral antigen is an influenza virus polypeptide other than ahemagglutinin polypeptide. In other embodiments, the viral antigen is aninfluenza virus hemagglutinin polypeptide.

Any anti-bacterial agents known to one of skill in the art may used incombination with an active compound (e.g, a chimeric influenzahemagglutinin (HA) polypeptide described herein, a nucleic acid encodingsuch a polypeptide, a vector (e.g., a viral vector, or a bacteria)containing or expressing such a polypeptide, cells stimulated with sucha polypeptide) or pharmaceutical composition described herein.Non-limiting examples of anti-bacterial agents include Amikacin,Amoxicillin, Amoxicillin-clavulanic acid, Amphothericin-B, Ampicillin,Ampicllin-sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin,Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin,Cefalothin, Cefazolin, Cefdinir, Cefepime, Cefixime, Cefmenoxime,Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefoxitin, Cefpirome,Cefpodoxime, Cefpodoxime-clavulanic acid, Cefpodoxime-sulbactam,Cefprozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole,Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin,Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin,Cotrimoxazol (Trimthoprim/sulphamethoxazole), Dalbavancin,Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin,Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin,Flucloxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid,Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem,Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin,Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem,Metronidazole, Meziocillin, Mezlocillin-sulbactam, Minocycline,Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin,Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin,Penicillin V, Piperacillin, Piperacillin-sulbactam,Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfloxacin,Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazole,Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyklin,Ticarcillin, Ticarcillin-clavulanic acid, Tigecycline, Tobramycin,Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin, andVoriconazole.

In some embodiments, a combination therapy comprises active immunizationwith a chimeric influenza hemagglutinin (HA) polypeptide describedherein, or one or more expression vectors described herein and passiveimmunization with one or more neutralizing antibodies generated and/oridentified using a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein. In some embodiments, a combination therapy comprisesimmunization with one or more expression vectors described herein andadministration of cells (e.g., by adoptive transfer) stimulated with achimeric influenza hemagglutinin (HA) polypeptide described herein.

In some embodiments, a combination therapy comprises administration oftwo or more different expression vectors described herein.

In some embodiments, a combination therapy comprises active immunizationwith an active compound (e.g, a chimeric influenza hemagglutinin (HA)polypeptide described herein, a nucleic acid encoding such apolypeptide, a vector (e.g., a viral vector, or a bacteria) containingor expressing such a polypeptide, cells stimulated with such apolypeptide) that induces an immune response to one, two, three, or moreHA subtypes in one HA group (e.g., Group 1) in combination with anactive compound (e.g, a chimeric influenza hemagglutinin (HA)polypeptide described herein, a nucleic acid encoding such apolypeptide, a vector (e.g., a viral vector, or a bacteria) containingor expressing such a polypeptide, cells stimulated with such apolypeptide) that induces an immune response to one, two, three, or moreHA subtypes in the other HA group (e.g., Group 2).

In some embodiments, a combination therapy comprises active immunizationwith two or more chimeric influenza hemagglutinin (HA) polypeptidesdescribed herein.

5.11.2 Patient Populations

In certain embodiments, an active compound (e.g, a chimeric influenzahemagglutinin (HA) polypeptide described herein, a nucleic acid encodingsuch a polypeptide, a vector (e.g., a viral vector, or a bacteria)containing or expressing such a polypeptide, cells stimulated with sucha polypeptide) or composition described herein may be administered to anaïve subject, i.e., a subject that does not have a disease caused byinfluenza virus infection or has not been and is not currently infectedwith an influenza virus infection. In one embodiment, an active compoundor composition described herein is administered to a naïve subject thatis at risk of acquiring an influenza virus infection. In one embodiment,an active compound or composition described herein is administered to asubject that does not have a disease caused by the specific influenzavirus, or has not been and is not infected with the specific influenzavirus to which the chimeric influenza hemagglutinin (HA) polypeptidedescribed herein induces an immune response. An active compound orcomposition described herein may also be administered to a subject thatis and/or has been infected with the influenza virus or another type,subtype or strain of the influenza virus to which the chimeric influenzahemagglutinin (HA) polypeptide induces an immune response.

In certain embodiments, an active compound (e.g, a chimeric influenzahemagglutinin (HA) polypeptide described herein, a nucleic acid encodingsuch a polypeptide, a vector (e.g., a viral vector, or a bacteria)containing or expressing such a polypeptide, cells stimulated with sucha polypeptide) or composition described herein is administered to apatient who has been diagnosed with an influenza virus infection. Insome embodiments, an active compound or composition described herein isadministered to a patient infected with an influenza virus beforesymptoms manifest or symptoms become severe (e.g., before the patientrequires hospitalization). In some embodiments, an active compound orcomposition described herein is administered to a patient that isinfected with or has been diagnosed with a different type of influenzavirus than that of the influenza virus from which the head domain of thechimeric influenza hemagglutinin (HA) polypeptide of the active compoundor composition was derived.

In certain embodiments, an active compound (e.g, a chimeric influenzahemagglutinin (HA) polypeptide described herein, a nucleic acid encodingsuch a polypeptide, a vector (e.g., a viral vector, or a bacteria)containing or expressing such a polypeptide, cells stimulated with sucha polypeptide) or composition described herein is administered to apatient that may be or is infected with an influenza virus that belongsto the same HA group as that of the head domain of the chimericinfluenza hemagglutinin (HA) polypeptide. In certain embodiments, anactive compound or composition described herein is administered to apatient that may be or is infected with an influenza virus of the samesubtype as that of the head domain of the chimeric influenzahemagglutinin (HA) polypeptide.

In some embodiments, a subject to be administered an active compound(e.g, a chimeric influenza hemagglutinin (HA) polypeptide describedherein, a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or compositiondescribed herein is an animal. In certain embodiments, the animal is abird. In certain embodiments, the animal is a canine. In certainembodiments, the animal is a feline. In certain embodiments, the animalis a horse. In certain embodiments, the animal is a cow. In certainembodiments, the animal is a mammal, e.g., a horse, swine, mouse, orprimate, preferably a human.

In certain embodiments, a subject to be administered an active compound(e.g, a chimeric influenza hemagglutinin (HA) polypeptide describedherein, a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or compositiondescribed herein is a human adult. In certain embodiments, a subject tobe administered an active compound or composition described herein is ahuman adult more than 50 years old. In certain embodiments, a subject tobe administered an active compound or composition described herein is anelderly human subject.

In certain embodiments, a subject to be administered an active compound(e.g, a chimeric influenza hemagglutinin (HA) polypeptide describedherein, a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or compositiondescribed herein is a human child. In certain embodiments, a subject tobe administered an active compound or composition described herein is ahuman infant. In certain embodiments, a subject to whom an activecompound or composition described herein is administered is not aninfant of less than 6 months old. In a specific embodiment, a subject tobe administered an active compound or composition described herein is ahuman 2 years old or younger. In another specific embodiment, a subjectto be administered an active compound or composition described herein isa human 5 years old or younger. In another specific embodiment, asubject to be administered an active compound or composition describedherein is a human 1-5 years old.

In specific embodiments, a subject to be administered an active compound(i.e., a chimeric influenza hemagglutinin (HA) polypeptide describedherein, a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or compositiondescribed herein is any infant or child more than 6 months of age andany adult over 50 years of age. In other embodiments, the subject is anindividual who is pregnant. In another embodiment, the subject is anindividual who may or will be pregnant during the influenza season(e.g., generally, November to April in the Northern hemisphere). Inspecific embodiments, a subject to be administered an active compound orcomposition described herein is a woman who has given birth 1, 2, 3, 4,5, 6, 7, or 8 weeks earlier.

In some embodiments, the human subject to be administered an activecompound (e.g, a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein, a nucleic acid encoding such a polypeptide, a vector(e.g., a viral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or compositiondescribed herein is any individual at increased risk of influenza virusinfection or disease resulting from influenza virus infection (e.g., animmunocompromised or immunodeficient individual). In some embodiments,the human subject to be administered an active compound or compositiondescribed herein is any individual in close contact with an individualwith increased risk of influenza virus infection or disease resultingfrom influenza virus infection (e.g., immunocompromised orimmunosuppressed individuals).

In some embodiments, the human subject to be administered an activecompound (e.g, a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein, a nucleic acid encoding such a polypeptide, a vector(e.g., a viral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or compositiondescribed herein is an individual affected by any condition thatincreases susceptibility to influenza virus infection or complicationsor disease resulting from influenza virus infection. In otherembodiments, an active compound or composition described herein isadministered to a subject in which an influenza virus infection has thepotential to increase complications of another condition that theindividual is affected by, or for which they are at risk. In particularembodiments, such conditions that increase susceptibility to influenzavirus complications or for which influenza virus increases complicationsassociated with the condition are, e.g., conditions that affect thelung, such as cystic fibrosis, chronic obstructive pulmonary disease(COPD), emphysema, asthma, or bacterial infections (e.g., infectionscaused by Haemophilus influenzae, Streptococcus pneumoniae, Legionellapneumophila, and Chlamydia trachomatus); cardiovascular disease (e.g.,congenital heart disease, congestive heart failure, and coronary arterydisease); endocrine disorders (e.g., diabetes), neurological andneuron-developmental conditions (e.g., disorders of the brain, thespinal cord, the peripheral nerve, and muscle (such as cerebral palsy,epilepsy (seizure disorders), stroke, intellectual disability (e,g,mental retardation), muscular dystrophy, and spinal cord injury)).

In some embodiments, the human subject to be administered an activecompound (e.g, a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein, a nucleic acid encoding such a polypeptide, a vector(e.g., a viral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or compositiondescribed herein is an individual that resides in a group home, such asa nursing home. In some embodiments, the human subject to beadministered an active compound or composition described herein worksin, or spends a significant amount of time in, a group home, e.g., anursing home. In some embodiments, the human subject to be administeredan active compound or composition described herein is a health careworker (e.g., a doctor or nurse). In some embodiments, the human subjectto be administered an active compound or composition described herein isa smoker. In a specific embodiment, the human subject to be administeredan active compound or composition described herein is immunocompromisedor immunosuppressed.

In addition, subjects at increased risk of developing complications frominfluenza who may be administered an active compound (e.g, a chimericinfluenza hemagglutinin (HA) polypeptide described herein, a nucleicacid encoding such a polypeptide, a vector (e.g., a viral vector, or abacteria) containing or expressing such a polypeptide, cells stimulatedwith such a polypeptide) or composition described herein include: anyindividual who can transmit influenza viruses to those at high risk forcomplications, such as, e.g., members of households with high-riskindividuals, including households that will include infants younger than6 months, individuals coming into contact with infants less than 6months of age, or individuals who will come into contact withindividuals who live in nursing homes or other long-term carefacilities; individuals with long-term disorders of the lungs, heart, orcirculation; individuals with metabolic diseases (e.g., diabetes);individuals with kidney disorders; individuals with blood disorders(including anemia or sickle cell disease); individuals with weakenedimmune systems (including immunosuppression caused by medications,malignancies such as cancer, organ transplant, or HIV infection);children who receive long-term aspirin therapy (and therefore have ahigher chance of developing Reye syndrome if infected with influenza).

In other embodiments, subjects for administration of an active compound(e.g, a chimeric influenza hemagglutinin (HA) polypeptide describedherein, a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or compositiondescribed herein include healthy individuals six months of age or older,who: plan to travel to foreign countries and areas where flu outbreaksmay be occurring, such, e.g., as the tropics and the Southern Hemispherefrom April through September; travel as a part of large organizedtourist groups that may include persons from areas of the world whereinfluenza viruses are circulating; attend school or college and residein dormitories, or reside in institutional settings; or wish to reducetheir risk of becoming ill with influenza.

In some embodiments, a subject for whom administration of an activecompound (e.g, a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein, a nucleic acid encoding such a polypeptide, a vector(e.g., a viral vector, or a bacteria) containing or expressing such apolypeptide, cells stimulated with such a polypeptide) or compositiondescribed herein is contraindicated include any individual for whominfluenza vaccination is contraindicated, such as: infants younger thansix months of age; and individuals who have had an anaphylactic reaction(allergic reactions that cause difficulty breathing, which is oftenfollowed by shock) to eggs, egg products, or other components used inthe production of the immunogenic formulation. In certain embodiments,when administration of an active compound or composition describedherein is contraindicated due to one or more components used in theproduction of the immunogenic formulation (e.g., due to the presence ofegg or egg products), the active compound or composition may be producedin a manner that does not include the component that causes theadministration of an active compound or composition to becontraindicated (e.g., the active compound or composition may beproduced without the use of eggs or egg products).

In some embodiments, it may be advisable not to administer a live virusvaccine to one or more of the following patient populations: elderlyhumans; infants younger than 6 months old; pregnant individuals; infantsunder the age of 1 years old; children under the age of 2 years old;children under the age of 3 years old; children under the age of 4 yearsold; children under the age of 5 years old; adults under the age of 20years old; adults under the age of 25 years old; adults under the age of30 years old; adults under the age of 35 years old; adults under the ageof 40 years old; adults under the age of 45 years old; adults under theage of 50 years old; elderly humans over the age of 70 years old;elderly humans over the age of 75 years old; elderly humans over the ageof 80 years old; elderly humans over the age of 85 years old; elderlyhumans over the age of 90 years old; elderly humans over the age of 95years old; children and adolescents (2-17 years of age) receivingaspirin or aspirin-containing medications, because of the complicationsassociated with aspirin and wild-type influenza virus infections in thisage group; individuals with a history of asthma or other reactive airwaydiseases; individuals with chronic underlying medical conditions thatmay predispose them to severe influenza infections; individuals with ahistory of Guillain-Barre syndrome; individuals with acute seriousillness with fever; or individuals who are moderately or severely ill.For such individuals, administration of inactivated virus vaccines,split virus vaccines, subunit vaccines, virosomes, virus-like particlesor the non-viral vectors described herein may be preferred. In certainembodiments, subjects preferably administered a live virus vaccine mayinclude healthy children and adolescents, ages 2-17 years, and healthyadults, ages 18-49.

In certain embodiments, an immunogenic formulation comprising a livevirus vector is not given concurrently with other live-virus vaccines.

5.12 Modes of Administration 5.12.1 Routes of Delivery

An active compound (e.g, a chimeric influenza hemagglutinin (HA)polypeptide described herein, a nucleic acid encoding such apolypeptide, a vector (e.g., a viral vector, or a bacteria) containingor expressing such a polypeptide, cells stimulated with such apolypeptide) or composition described herein may be delivered to asubject by a variety of routes. These include, but are not limited to,intranasal, intratracheal, oral, intradermal, intramuscular,intraperitoneal, transdermal, intravenous, conjunctival and subcutaneousroutes. In some embodiments, a composition is formulated for topicaladministration, for example, for application to the skin. In specificembodiments, the route of administration is nasal, e.g., as part of anasal spray. In certain embodiments, a composition is formulated forintramuscular administration. In some embodiments, a composition isformulated for subcutaneous administration. In certain embodiments, acomposition is not formulated for administration by injection. Inspecific embodiments for live virus vaccines, the vaccine is formulatedfor administration by a route other than injection.

In cases where the antigen is a viral vector, a virus-like particlevector, or a bacterial vector, for example, it may be preferable tointroduce an immunogenic composition via the natural route of infectionof the backbone virus or bacteria from which the vector was derived.Alternatively, it may be preferable to introduce a chimeric influenzahemagglutinin (HA) polypeptide described herein via the natural route ofinfection of the influenza virus from which polypeptide is derived. Theability of an antigen, particularly a viral vector, to induce a vigoroussecretory and cellular immune response can be used advantageously. Forexample, infection of the respiratory tract by a viral vector may inducea strong secretory immune response, for example in the urogenitalsystem, with concomitant protection against an influenza virus. Inaddition, in a preferred embodiment it may be desirable to introduce thepharmaceutical compositions into the lungs by any suitable route.Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent for useas a spray.

In a specific embodiment, a subunit vaccine is administeredintramuscularly. In another embodiment, a live influenza virus vaccineis administered intranasally. In another embodiment, an inactivatedinfluenza virus vaccine, or a split influenza virus vaccine isadministered intramuscularly. In another embodiment, a virus-likeparticle or composition thereof is administered intramuscularly.

In some embodiments, cells stimulated with a fl chimeric influenzahemagglutinin (HA) polypeptide described herein in vitro may beintroduced (or re-introduced) into a subject using techniques known toone of skill in the art. In some embodiments, the cells can beintroduced into the dermis, under the dermis, or into the peripheralblood stream. In some embodiments, the cells introduced into a subjectare preferably cells derived from that subject, to avoid an adverseimmune response. In other embodiments, cells also can be used that arederived from a donor host having a similar immune background. Othercells also can be used, including those designed to avoid an adverseimmunogenic response.

5.12.2 Dosage and Frequency of Administration

The amount of an active compound (e.g, a chimeric influenzahemagglutinin (HA) polypeptide described herein, a nucleic acid encodingsuch a polypeptide, a vector (e.g., a viral vector, or a bacteria)containing or expressing such a polypeptide, cells stimulated with sucha polypeptide) or composition which will be effective in the treatmentand/or prevention of an influenza virus infection or an influenza virusdisease will depend on the nature of the disease, and can be determinedby standard clinical techniques.

The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of the infection ordisease caused by it, and should be decided according to the judgment ofthe practitioner and each subject's circumstances. For example,effective doses may also vary depending upon means of administration,target site, physiological state of the patient (including age, bodyweight, health), whether the patient is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the patient is a human but nonhuman mammalsincluding transgenic mammals can also be treated. Treatment dosages areoptimally titrated to optimize safety and efficacy.

In certain embodiments, an in vitro assay is employed to help identifyoptimal dosage ranges. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

Exemplary doses for nucleic acids encoding a chimeric influenzahemagglutinin (HA) polypeptide described herein range from about 10 ngto 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg nucleic acid,e.g., DNA, per patient.

In certain embodiments, exemplary doses for a chimeric influenzahemagglutinin (HA) polypeptide described herein (e.g., as provided insplit virus vaccines and subunit vaccines) range from about 0.5 μg to1.0 μg, 1.0 μg to 2.0 μg, 2.0 μg to 5.0 μg, 5.0 μg to 10 μg, 15 μg to 25μg, 25 μg to 50 μg, 50 μg to 100 μg, 100 μg to 500 μg, or 500 μg to 1.0mg, of chimeric influenza hemagglutinin (HA) polypeptide per kilogram ofthe patient. In other embodiments, exemplary doses for a chimericinfluenza hemagglutinin (HA) polypeptide described herein range fromabout 0.5 μg to 1.0 μg, 1.0 μg to 2.0 μg, 2.0 μg to 5.0 μg, 5.0 μg to 10μg, 15 μg to 25 μg, 25 μg to 50 μg, 50 μg to 100 μg, 100 μg to 500 μg,250 μg to 500 μg, 500 μg to 1.0 mg, or 750 μg to 1 mg of chimericinfluenza hemagglutinin (HA) polypeptide per dose, and can beadministered to a subject once, twice, three times or more than threetimes with intervals as often as needed.

Doses for infectious viral vectors may vary from 10-100, or more,virions per dose. In some embodiments, suitable dosages of a virusvector are 10², 5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶,10⁷, 5×10⁷, 10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or10¹² pfu, and can be administered to a subject once, twice, three ormore times with intervals as often as needed.

In certain embodiments, exemplary doses for VLPs range from about 0.01μg to about 100 mg, about 0.1 μg to about 100 mg, about 5 μg to about100 mg, about 15 μg to about 50 mg, about 15 μg to about 25 mg, about 15μg to about 10 mg, about 15 μg to about 5 mg, about 15 μg to about 1 mg,about 15 μg to about 100 μg, about 15 μg to about 75 μg, about 5 μg toabout 50 μg, about 10 μg to about 50 μg, about 15 μg to about 45 μg,about 20 μg to about 40 μg, or about 25 to about 35 μg per kilogram ofthe patient.

In one embodiment, an inactivated vaccine is formulated such that itcontains about 5 μg to about 50 μg, about 10 μg to about 50 μg, about 15μg to about 100 μg, about 15 μg to about 75 μg, about 15 μg to about 50μg, about 15 μg to about 30 μg, about 20 μg to about 50 μg, about 25 μgto about 40 μg, about 25 μg to about 35 μg of a chimeric influenzahemagglutinin (HA) polypeptide.

In certain embodiments, an active compound i.e., a chimeric influenzahemagglutinin (HA) polypeptide described herein, a nucleic acid encodingsuch a polypeptide, a vector (e.g., a viral vector, or a bacteria)containing or expressing such a polypeptide, cells stimulated with sucha polypeptide, or composition is administered to a subject once as asingle dose. For example, subjects (e.g., older subjects) that havepreviously been exposed to influenza may not require multipleadministrations of an active compound described herein or compositionthereof, but, rather may be sufficiently vaccinated by a singleimmunization with an active compound described herein or compositionthereof. Alternatively, subjects (e.g., older subjects) that havepreviously been exposed to influenza may require multipleadministrations of an active compound described herein or compositionthereof, but may require less of such administrations than required bynaive subjects (i.e., subjects not previously exposed to influenza) tobecome sufficiently vaccinated. Accordingly, in certain embodiments,naive subjects may require a first immunization (e.g., priming) with anactive compound described herein or composition thereof followed by one,two, or more additional immunizations (e.g., boostings) with an activecompound described herein or composition thereof.

In a specific embodiment, where a subject is administered more than oneactive compounds described herein or compositions thereof in succession(e.g., as part of an immunization regimen), the chimeric influenzahemagglutinin (HA) polypeptides of the active compounds or compositionsthereof used in the successive administrations (i.e., theadministrations that take place after the first administration) differfrom the chimeric influenza hemagglutinin (HA) polypeptide of the activecompound or composition thereof used in the first administration. Inspecific embodiments, the chimeric influenza hemagglutinin (HA)polypeptides of the active compounds or compositions thereof used in thesuccessive administrations comprise a different globular head domainthan the chimeric influenza hemagglutinin (HA) polypeptide of the activecompound or composition thereof used in the first administration butcomprise the same HA stem domain as the chimeric influenza hemagglutinin(HA) polypeptide of the active compound or composition thereof used inthe first administration but comprise the same HA stem domain. Inspecific embodiments, the chimeric influenza hemagglutinin (HA)polypeptides of the active compounds or compositions thereof used in thesuccessive administrations comprise a different globular head domain anda different HA stem domain than the chimeric influenza hemagglutinin(HA) polypeptide of the active compound or composition thereof used inthe first administration.

In certain embodiments, an active compound or composition (e.g.,composition comprising an active compound) is administered to a subjectas a single dose followed by a second dose 3 to 6 weeks later. Incertain embodiments, an active compound or composition is administeredto a subject as a single dose followed by a second dose 3 to 6 weekslater, which is followed by administration of a third dose 3 to 6 weekslater. In certain embodiments, the second and/or third administrationsmay utilize a different active compound or composition. In specificembodiments, the globular head domain of each chimeric influenzahemagglutinin (HA) polypeptide in the active compounds or compositionsadministered are different from one another, e.g., the first and second,or the first, second, and third administrations each use a differentactive compound or composition, wherein at least the globular headdomain of the chimeric influenza hemagglutinin (HA) polypeptide in eachactive compound or composition administered differs. In accordance withthese embodiments, booster inoculations may be administered to thesubject at, e.g., 3 to 6 month, 6 to 9 month, or 6 to 12 month intervalsfollowing the second inoculation. In certain embodiments, the boosterinoculations may utilize a different active compound or composition. Incertain embodiments, the first (priming) administration comprises afull-length hemagglutinin or fragment thereof (or a nucleic acidencoding the same) and the second (booster) administration comprisesadministration of a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein (or a nucleic acid encoding the same, a VLP comprisingthe same, or a virus or bacteria expressing the same). In someembodiments, the administration of the same active compound orcomposition may be repeated and the administrations may be separated byat least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45days, 2 months, 75 days, 3 months, or at least 6 months. In certainembodiments, an active compound or composition is administered to asubject as a single dose once per year.

In specific embodiments for administration to children, two doses of anactive compound (e.g, a chimeric influenza hemagglutinin (HA)polypeptide described herein, a nucleic acid encoding such apolypeptide, a vector (e.g., a viral vector, or a bacteria) containingor expressing such a polypeptide, cells stimulated with such apolypeptide) or composition, given at least one month apart, areadministered to a child. In specific embodiments for administration toadults, a single dose is given. In another embodiment, two doses of anactive compound or composition, given at least one month apart, areadministered to an adult. In another embodiment, a young child (sixmonths to nine years old) may be administered an active compound orcomposition for the first time in two doses given one month apart. In aparticular embodiment, a child who received only one dose in their firstyear of vaccination should receive two doses in the following year. Insome embodiments, two doses administered 4 weeks apart are preferred forchildren 2-8 years of age who are administered an influenza vaccine,e.g., an immunogenic formulation described herein, for the first time.In certain embodiments, for children 6-35 months of age, a half dose(0.25 ml) may be preferred, in contrast to 0.5 ml which may be preferredfor subjects over three years of age.

In a specific embodiment, for administration to human infants, two dosesof a chimeric influenza hemagglutinin (HA) polypeptide described hereinor a composition thereof and/or one or more of the nucleic acids,vectors, VLPs, or virosomes described herein, are administered to aninfant, wherein the influenza virus hemagglutinin head domain of thechimeric influenza hemagglutinin (HA) polypeptide used in the first doseis from a different strain or subtype than the influenza virushemagglutinin head domain of the chimeric influenza hemagglutinin (HA)polypeptide used in the second dose. The first and secondadministrations may be separated by at least 1 day, 2 days, 3 days, 5days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months,or at least 6 months.

In a specific embodiment, for administration to human infants, threedoses of a chimeric influenza hemagglutinin (HA) polypeptide describedherein described herein or a composition thereof and/or one or more ofthe nucleic acids, vectors, VLPs, or virosomes described herein, areadministered to an infant, wherein the influenza virus hemagglutininhead domains of the chimeric influenza hemagglutinin (HA) polypeptidesused in the first, second, and third doses are from different strains orsubtypes of influenza virus. The first, second, and thirdadministrations may be separated by at least 1 day, 2 days, 3 days, 5days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months,or at least 6 months.

In particular embodiments, an active compound (e.g, a chimeric influenzahemagglutinin (HA) polypeptide described herein, a nucleic acid encodingsuch a polypeptide, a vector (e.g., a viral vector, or a bacteria)containing or expressing such a polypeptide, cells stimulated with sucha polypeptide) or composition is administered to a subject in the fallor winter, i.e., prior to or during the influenza season in eachhemisphere. In one embodiment, children are administered their firstdose early in the season, e.g., late September or early October in theNorthern hemisphere, so that the second dose can be given prior to thepeak of the influenza season.

For passive immunization with an antibody (e.g., an antibody generatedand/or identified using a chimeric influenza hemagglutinin (HA)polypeptide described herein), the dosage ranges from about 0.0001 to100 mg/kg, and more usually 0.01 to 5 mg/kg, of the patient body weight.For example, dosages can be 1 mg/kg body weight or 10 mg/kg body weightor within the range of 1-10 mg/kg or in other words, 70 mg or 700 mg orwithin the range of 70-700 mg, respectively, for a 70 kg patient. Anexemplary treatment regime entails administration once per every twoweeks or once a month or once every 3 to 6 months for a period of oneyear or over several years, or over several year-intervals. In somemethods, two or more monoclonal antibodies with different bindingspecificities are administered simultaneously, in which case the dosageof each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody tothe chimeric influenza hemagglutinin (HA) polypeptide in the patient.

5.13 Biological Assays 5.13.1 Assays for Testing Activity of ChimericInfluenza Virus Hemagglutinin Polypeptides

Assays for testing the expression of a chimeric influenza hemagglutinin(HA) polypeptide in a vector disclosed herein may be conducted using anyassay known in the art. For example, an assay for incorporation into aviral vector comprises growing the virus, purifying the viral particlesby centrifugation through a sucrose cushion, and subsequent analysis forchimeric influenza hemagglutinin (HA) polypeptide expression by animmunoassay, such as Western blotting, using methods well known in theart. Methods for determining whether a hemagglutinin polypeptide ischimeric are known to those of skill in the art and described herein.

In one embodiment, a chimeric influenza hemagglutinin (HA) polypeptidedisclosed herein is assayed for proper folding and functionality bytesting its ability to bind specifically to a neutralizing antibodydirected to an influenza virus hemagglutinin polypeptide, such as thestalk region of the polypeptide, using any assay for antibody-antigeninteraction known in the art. Neutralizing antibodies for use in suchassays include, for example, the neutralizing antibodies described inEkiert et al., 2009, Science Express, 26 Feb. 2009; Kashyap et al.,2008, Proc Natl Acad Sci USA 105: 5986-5991; Sui et al. 2009, NatureStructural and Molecular Biology, 16:265-273; Wang et al., 2010, PLOSPathogens 6(2):1-9; U.S. Pat. Nos. 5,589,174, 5,631,350, 6,337,070, and6,720,409; International Application No. PCT/US2007/068983 published asInternational Publication No. WO 2007/134237; International ApplicationNo. PCT/US2008/075998 published as International Publication No. WO2009/036157; International Application No. PCT/EP2007/059356 publishedas International Publication No. WO 2008/028946; and InternationalApplication No. PCT/US2008/085876 published as International PublicationNo. WO 2009/079259. These antibodies include CR6261, CR6325, CR6329,CR6307, CR6323, 2A, D7, D8, F10, G17, H40, A66, D80, E88, E90, H98, C179(FERM BP-4517), AI3C (FERM BP-4516), among others.

In another embodiment, a chimeric influenza hemagglutinin (HA)polypeptide disclosed herein is assayed for proper folding bydetermination of the structure or conformation of the chimeric influenzahemagglutinin (HA) polypeptide using any method known in the art suchas, e.g., NMR, X-ray crystallographic methods, or secondary structureprediction methods, e.g., circular dichroism.

5.13.2 Assays for Testing Activity of Antibodies Generated UsingChimeric Influenza Virus Hemagglutinin Polypeptides

Antibodies described herein may be characterized in a variety of waysknown to one of skill in the art (e.g. ELISA, Surface Plasmon resonancedisplay (BIAcore), Western blot, immunofluorescence, immunostainingand/or microneutralization assays). In some embodiments, antibodies areassayed for the ability to specifically bind to a chimeric influenzahemagglutinin (HA) polypeptide, or a vector comprising said polypeptide.Such an assay may be performed in solution (e.g., Houghten, 1992,Bio/Techniques 13:412 421), on beads (Lam, 1991, Nature 354:82 84), onchips (Fodor, 1993, Nature 364:555 556), on bacteria (U.S. Pat. No.5,223,409), on spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and5,223,409), on plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA89:1865 1869) or on phage (Scott and Smith, 1990, Science 249:386 390;Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378 6382; andFelici, 1991, J. Mol. Biol. 222:301 310) (each of these references isincorporated herein in its entirety by reference).

Specific binding of an antibody to the chimeric influenza hemagglutinin(HA) polypeptide and cross-reactivity with other antigens can beassessed by any method known in the art. Immunoassays which can be usedto analyze specific binding and cross-reactivity include, but are notlimited to, competitive and non-competitive assay systems usingtechniques such as western blots, radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds.,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York, which is incorporated by reference herein in itsentirety).

The binding affinity of an antibody to a chimeric influenzahemagglutinin (HA) polypeptide and the off-rate of an antibody-antigeninteraction can be determined by competitive binding assays. One exampleof a competitive binding assay is a radioimmunoassay comprising theincubation of labeled antigen (e.g., ³H or ¹²⁵I) with the antibody ofinterest in the presence of increasing amounts of unlabeled antigen, andthe detection of the antibody bound to the labeled antigen. The affinityof the antibody for a chimeric influenza hemagglutinin (HA) polypeptideand the binding off-rates can be determined from the data by Scatchardplot analysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, a chimeric influenzahemagglutinin (HA) polypeptide is incubated with the test antibodyconjugated to a labeled compound (e.g., ³H or ¹²⁵I) in the presence ofincreasing amounts of an unlabeled second antibody.

In certain embodiments, antibody binding affinity and rate constants aremeasured using the KinExA 3000 System (Sapidyne Instruments, Boise,Id.). In some embodiments, surface plasmon resonance (e.g., BIAcorekinetic) analysis is used to determine the binding on and off rates ofthe antibodies to an influenza virus hemagglutinin polypeptide. BIAcorekinetic analysis comprises analyzing the binding and dissociation of achimeric influenza hemagglutinin (HA) polypeptide from chips withimmobilized antibodies to chimeric influenza hemagglutinin (HA)polypeptide on their surface. A typical BIAcore kinetic study involvesthe injection of 250 μL of an antibody reagent (mAb, Fab) at varyingconcentration in HBS buffer containing 0.005% Tween-20 over a sensorchip surface, onto which has been immobilized the chimeric influenzahemagglutinin (HA) polypeptide. The flow rate is maintained constant at75 μL/min. Dissociation data is collected for 15 min or longer asnecessary. Following each injection/dissociation cycle, the boundantibody is removed from the influenza virus hemagglutinin polypeptidesurface using brief, 1 min pulses of dilute acid, typically 10-100 mMHCl, though other regenerants are employed as the circumstances warrant.More specifically, for measurement of the rates of association, k_(on),and dissociation, k_(off), the polypeptide is directly immobilized ontothe sensor chip surface through the use of standard amine couplingchemistries, namely the EDC/NHS method(EDC=N-diethylaminopropyl)-carbodiimide). Briefly, a 5-100 nM solutionof the polypeptide in 10 mM NaOAc, pH 4 or pH 5 is prepared and passedover the EDC/NHS-activated surface until approximately 30-50 RU's worthof polypeptide are immobilized. Following this, the unreacted activeesters are “capped” off with an injection of 1M Et-NH₂. A blank surface,containing no polypeptide, is prepared under identical immobilizationconditions for reference purposes. Once an appropriate surface has beenprepared, a suitable dilution series of each one of the antibodyreagents is prepared in HBS/Tween-20, and passed over both thepolypeptide and reference cell surfaces, which are connected in series.The range of antibody concentrations that are prepared varies, dependingon what the equilibrium binding constant, K_(D), is estimated to be. Asdescribed above, the bound antibody is removed after eachinjection/dissociation cycle using an appropriate regenerant.

The neutralizing activity of an antibody can be determined utilizing anyassay known to one skilled in the art. Antibodies described herein canbe assayed for their ability to inhibit the binding of an influenzavirus, or any other composition comprising a chimeric influenzahemagglutinin (HA) polypeptide (e.g., a VLP, liposome, or detergentextract), to its host cell receptor (i.e., sialic acid) using techniquesknown to those of skill in the art. For example, cells expressinginfluenza virus receptors can be contacted with a composition comprisinga chimeric influenza hemagglutinin (HA) polypeptide described herein inthe presence or absence of the antibody and the ability of the antibodyto inhibit the antigen's binding can measured by, for example, flowcytometry or a scintillation assay. The composition comprising achimeric influenza hemagglutinin (HA) polypeptide described herein orthe antibody can be labeled with a detectable compound such as aradioactive label (e.g., ³²P, ³⁵S, and ¹²⁵I) or a fluorescent label(e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine) toenable detection of an interaction between the composition comprising achimeric influenza hemagglutinin (HA) polypeptide described herein and acell receptor. Alternatively, the ability of antibodies to inhibit achimeric influenza hemagglutinin (HA) polypeptide described herein frombinding to its receptor can be determined in cell-free assays. Forexample, a composition comprising a chimeric influenza hemagglutinin(HA) polypeptide described herein can be contacted with an antibody andthe ability of the antibody to inhibit the composition comprising thechimeric influenza hemagglutinin (HA) polypeptide from binding to a cellreceptor can be determined. In a specific embodiment, the antibody isimmobilized on a solid support and the composition comprising aninfluenza virus hemagglutinin polypeptide is labeled with a detectablecompound. Alternatively, a composition comprising a chimeric influenzahemagglutinin (HA) polypeptide described herein is immobilized on asolid support and the antibody is labeled with a detectable compound. Incertain embodiments, the ability of an antibody to inhibit a chimericinfluenza hemagglutinin (HA) polypeptide described herein from bindingto a cell receptor is determined by assessing the percentage of bindinginhibition of the antibody relative to a control (e.g., an antibodyknown to inhibit the chimeric influenza hemagglutinin (HA) polypeptidefrom binding to the cell receptor).

In other embodiments, an antibody suitable for use in the methodsdescribed herein does not inhibit influenza virus receptor binding, yetis still found to be neutralizing in an assay described herein. In someembodiments, an antibody suitable for use in accordance with the methodsdescribed herein reduces or inhibits virus-host membrane fusion in anassay known in the art or described herein.

In one embodiment, virus-host membrane fusion is assayed in an in vitroassay using an influenza virus containing a reporter and a host cellcapable of being infected with the virus. An antibody inhibits fusion ifreporter activity is inhibited or reduced compared to a negative control(e.g., reporter activity in the presence of a control antibody or in theabsence of antibody).

In one embodiment, virus-host membrane fusion is detected using a modelsystem of cell fusion. In an exemplary cell fusion assay, cells (e.g.,HeLa cells) are transfected with a plasmid encoding a chimeric influenzahemagglutinin (HA) polypeptide described herein and contacted andexposed to a buffer that allows the chimeric influenza hemagglutinin(HA) polypeptide fusion function (e.g., pH 5.0 buffer) in the presenceof an antibody. An antibody is neutralizing if it reduces or inhibitssyncytia formation compared to a negative control (e.g., syncytiaformation in the presence of a control antibody or in the absence ofantibody).

In other embodiments, virus-host membrane fusion is assayed using an invitro liposome-based assay. In an exemplary assay, the host cellreceptor is reconstituted into liposomes containing one half of areporter. A chimeric influenza hemagglutinin (HA) polypeptide describedherein is reconstituted into another set of liposomes containing anotherhalf of a reporter. When the two liposome populations are mixedtogether, fusion is detected by reconstitution of the reporter, forexample, an enzymatic reaction that can be detected colorimetrically.The antibody inhibits fusion if reporter activity is reduced orinhibited compared to reporter activity in an assay conducted in theabsence of antibody or in the presence of a control antibody. In certainembodiments, the ability of an antibody to inhibit fusion is determinedby assessing the percentage of fusion in the presence of the antibodyrelative to the percentage of fusion in the presence a control.

5.13.3 Assays for Testing Activity of Stimulated Cells

Cells stimulated in accordance with the methods described herein may beanalyzed, for example, for integration, transcription and/or expressionof the polynucleotide or gene(s) of interest, the number of copies ofthe gene integrated, and the location of the integration. Such analysismay be carried out at any time and may be carried out by any methodsknown in the art. In other embodiments, successful stimulation of thetarget cell with a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein is determined by detecting production of neutralizingantibodies against the chimeric influenza hemagglutinin (HA) polypeptideusing methods known in the art or described herein.

In certain embodiments, subjects in which the stimulated cells, e.g.,DCs, are administered can be analyzed for location of the cells,expression of a vector-delivered polynucleotide or gene encoding thechimeric influenza hemagglutinin (HA) polypeptide, stimulation of animmune response (e.g., production of neutralizing antibodies against thechimeric influenza hemagglutinin (HA) polypeptide), and/or monitored forsymptoms associated with influenza virus infection or a diseaseassociated therewith by any methods known in the art or describedherein.

Reporter assays can be used to determine the specificity of thetargeting of the chimeric influenza hemagglutinin (HA) polypeptidedescribed herein. For example, a mixed population of bone marrow cellscan be obtained from a subject and cultured in vitro. The chimericinfluenza hemagglutinin (HA) polypeptide can be administered to themixed population of bone marrow cells, and expression of a reporter geneassociated with the fl chimeric influenza hemagglutinin (HA) polypeptidecan be assayed in the cultured cells. In some embodiments, at leastabout 50%, more preferably at least about 60%, 70%, 80% or 90%, stillmore preferably at least about 95% of stimulated cells in the mixed cellpopulation are dendritic cells.

5.13.4 Antiviral Activity Assays

Antibodies described herein or compositions thereof can be assessed invitro for antiviral activity. In one embodiment, the antibodies orcompositions thereof are tested in vitro for their effect on growth ofan influenza virus. Growth of influenza virus can be assessed by anymethod known in the art or described herein (e.g. in cell culture). In aspecific embodiment, cells are infected at a MOI of 0.0005 and 0.001,0.001 and 0.01, 0.01 and 0.1, 0.1 and 1, or 1 and 10, or a MOI of0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 or 10 and incubatedwith serum free media supplemented. Viral titers are determined in thesupernatant by hemagglutinin plaques or any other viral assay describedherein. Cells in which viral titers can be assessed include, but are notlimited to, EFK-2 cells, Vero cells, MDCK cells, primary human umbilicalvein endothelial cells (HUVEC), H292 human epithelial cell line and HeLacells. In vitro assays include those that measure altered viralreplication (as determined, e.g., by plaque formation) or the productionof viral proteins (as determined, e.g., by Western blot analysis) orviral RNAs (as determined, e.g., by RT-PCR or northern blot analysis) incultured cells in vitro using methods which are well known in the art ordescribed herein.

In one non-limiting example, a monolayer of the target mammalian cellline is infected with different amounts (e.g., multiplicity of 3 plaqueforming units (pfu) or 5 pfu) of virus (e.g., influenza) andsubsequently cultured in the presence or absence of various dilutions ofantibodies (e.g., 0.1 μg/ml, 1 μg/ml, 5 μg/ml, or 10 μg/ml). Infectedcultures are harvested 48 hours or 72 hours post infection and titeredby standard plaque assays known in the art on the appropriate targetcell line (e.g., Vero cells).

In a non-limiting example of a hemagglutination assay, cells arecontacted with an antibody and are concurrently or subsequently infectedwith the virus (e.g., at an MOI of 1) and the virus is incubated underconditions to permit virus replication (e.g., 20-24 hours). Theantibodies are preferably present throughout the course of infection.Viral replication and release of viral particles is then determined byhemagglutination assays using 0.5% chicken red blood cells. See, e.g.,Kashyap et al., PNAS USA 105: 5986-5991. In some embodiments, a compoundis considered an inhibitor of viral replication if it reduces viralreplication by at least 2 wells of HA, which equals approximately a 75%reduction in viral titer. In specific embodiments, an inhibitor reducesviral titer in this assay by 50% or more, by 55% or more, by 60% ormore, by 65% or more, by 70% or more, by 75% or more, by 80% or more, by85% or more, by 90% or more, or by 95% or more. In other specificembodiments an inhibitor results in a reduction of approximately 1 logor more, approximately 2 logs or more, approximately 3 logs or more,approximately 4 logs or more, approximately 5 logs or more,approximately 6 logs or more, approximately 7 logs or more,approximately 8 logs or more, approximately 9 logs or more,approximately 10 logs or more, 1 to 3 logs, 1 to 5 logs, 1 to 8 logs, 1to 9 logs, 2 to 10 logs, 2 to 5 logs, 2 to 7 logs, 2 logs to 8 logs, 2to 9 logs, 2 to 10 logs 3 to 5 logs, 3 to 7 logs, 3 to 8 logs, 3 to 9logs, 4 to 6 logs, 4 to 8 logs, 4 to 9 logs, 5 to 6 logs, 5 to 7 logs, 5to 8 logs, 5 to 9 logs, 6 to 7 logs, 6 to 8 logs, 6 to 9 logs, 7 to 8logs, 7 to 9 logs, or 8 to 9 logs in influenza virus titer in thesubject. The log-reduction in Influenza virus titer may be as comparedto a negative control, as compared to another treatment, or as comparedto the titer in the patient prior to antibody administration.

5.13.5 Cytotoxicity Assays

Many assays well-known in the art can be used to assess viability ofcells (infected or uninfected) or cell lines following exposure to anactive compound or a composition thereof and, thus, determine thecytotoxicity of the compound or composition. For example, cellproliferation can be assayed by measuring Bromodeoxyuridine (BrdU)incorporation (See, e.g., Hoshino et al., 1986, Int. J. Cancer 38, 369;Campana et al., 1988, J. Immunol. Meth. 107:79), (3H) thymidineincorporation (See, e.g., Chen, J., 1996, Oncogene 13:1395-403; Jeoung,J., 1995, J. Biol. Chem. 270:18367 73), by direct cell count, or bydetecting changes in transcription, translation or activity of knowngenes such as proto-oncogenes (e.g., fos, myc) or cell cycle markers(Rb, cdc2, cyclin A, D1, D2, D3, E, etc). The levels of such protein andmRNA and activity can be determined by any method well known in the art.For example, protein can be quantitated by known immunodiagnosticmethods such as ELISA, Western blotting or immunoprecipitation usingantibodies, including commercially available antibodies. mRNA can bequantitated using methods that are well known and routine in the art,for example, using northern analysis, RNase protection, or polymerasechain reaction in connection with reverse transcription. Cell viabilitycan be assessed by using trypan-blue staining or other cell death orviability markers known in the art. In a specific embodiment, the levelof cellular ATP is measured to determined cell viability.

In specific embodiments, cell viability is measured in three-day andseven-day periods using an assay standard in the art, such as theCellTiter-Glo Assay Kit (Promega) which measures levels of intracellularATP. A reduction in cellular ATP is indicative of a cytotoxic effect. Inanother specific embodiment, cell viability can be measured in theneutral red uptake assay. In other embodiments, visual observation formorphological changes may include enlargement, granularity, cells withragged edges, a filmy appearance, rounding, detachment from the surfaceof the well, or other changes. These changes are given a designation ofT (100% toxic), PVH (partially toxic—very heavy—80%), PH (partiallytoxic—heavy—60%), P (partially toxic—40%), Ps (partiallytoxic—slight—20%), or 0 (no toxicity—0%), conforming to the degree ofcytotoxicity seen. A 50% cell inhibitory (cytotoxic) concentration(IC₅₀) is determined by regression analysis of these data.

In a specific embodiment, the cells used in the cytotoxicity assay areanimal cells, including primary cells and cell lines. In someembodiments, the cells are human cells. In certain embodiments,cytotoxicity is assessed in one or more of the following cell lines:U937, a human monocyte cell line; primary peripheral blood mononuclearcells (PBMC); Huh7, a human hepatoblastoma cell line; 293T, a humanembryonic kidney cell line; and THP-1, monocytic cells. In certainembodiments, cytotoxicity is assessed in one or more of the followingcell lines: MDCK, MEF, Huh 7.5, Detroit, or human tracheobronchialepithelial (HTBE) cells.

Active compounds (e.g., chimeric influenza hemagglutinin (HA)polypeptides described herein) or compositions thereof can be tested forin vivo toxicity in animal models. For example, animal models, describedherein and/or others known in the art, used to test the activities ofactive compounds can also be used to determine the in vivo toxicity ofthese compounds. For example, animals are administered a range ofconcentrations of active compounds. Subsequently, the animals aremonitored over time for lethality, weight loss or failure to gainweight, and/or levels of serum markers that may be indicative of tissuedamage (e.g., creatine phosphokinase level as an indicator of generaltissue damage, level of glutamic oxalic acid transaminase or pyruvicacid transaminase as indicators for possible liver damage). These invivo assays may also be adapted to test the toxicity of variousadministration mode and/or regimen in addition to dosages.

The toxicity and/or efficacy of an active compound (e.g., chimericinfluenza hemagglutinin (HA) polypeptides described herein) can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. An active compound that exhibits large therapeutic indices ispreferred. While an active compound that exhibits toxic side effects maybe used, care should be taken to design a delivery system that targetssuch agents to the site of affected tissue in order to minimizepotential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of an active compound for use inhumans. The dosage of such agents lies preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyactive compound used in a method described herein, the effective dosecan be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high-performance liquid chromatography.Additional information concerning dosage determination is providedherein.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of the activecompounds and compositions described herein, for example, by measuringviral infection or a condition or symptoms associated therewith.

5.13.6 In Vivo Antiviral Activity

Active compounds (e.g., chimeric influenza hemagglutinin (HA)polypeptides described herein) and compositions thereof can be assayedin vivo for the desired therapeutic or prophylactic activity prior touse in humans. For example, in vivo assays can be used to determinewhether it is preferable to administer an active compound or compositionthereof and/or another therapy. For example, to assess the use of anactive compound or composition thereof to prevent an influenza virusdisease, the composition can be administered before the animal isinfected with influenza virus. Alternatively, or in addition, an activecompound or composition thereof can be administered to the animal at thesame time that the animal is infected with influenza virus. To assessthe use of an active compound or composition thereof to treat aninfluenza virus infection or disease associated therewith, the compoundor composition may be administered after infecting the animal withinfluenza virus. In a specific embodiment, an active compound orcomposition thereof is administered to the animal more than one time.

Active compounds (e.g., chimeric influenza hemagglutinin (HA)polypeptides described herein) and compositions thereof can be testedfor antiviral activity in animal model systems including, but are notlimited to, rats, mice, chicken, cows, monkeys, pigs, ferrets, goats,sheep, dogs, rabbits, guinea pigs, etc. In a specific embodiment, activecompounds and compositions thereof are tested in a mouse model system.Such model systems are widely used and well-known to the skilledartisan. In a specific embodiment, active compounds and compositionsthereof are tested in a mouse model system. Non-limiting examples ofanimal models for influenza virus are provided in this section.

In general, animals are infected with influenza virus and concurrentlyor subsequently treated with an active compound or composition thereof,or placebo. Alternatively, animals are treated with an active compoundor composition thereof or placebo and subsequently infected withinfluenza virus. Samples obtained from these animals (e.g., serum,urine, sputum, semen, saliva, plasma, or tissue sample) can be testedfor viral replication via well known methods in the art, e.g., thosethat measure altered viral titers (as determined, e.g., by plaqueformation), the production of viral proteins (as determined, e.g., byWestern blot, ELISA, or flow cytometry analysis) or the production ofviral nucleic acids (as determined, e.g., by RT-PCR or northern blotanalysis). For quantitation of virus in tissue samples, tissue samplesare homogenized in phosphate-buffered saline (PBS), and dilutions ofclarified homogenates are adsorbed for 1 hour at 37° C. onto monolayersof cells (e.g., Vero, CEF or MDCK cells). In other assays,histopathologic evaluations are performed after infection, preferablyevaluations of the organ(s) the virus is known to target for infection.Virus immunohistochemistry can be performed using a viral-specificmonoclonal antibody.

The effect of an active compound (e.g., chimeric influenza hemagglutinin(HA) polypeptides described herein) or composition thereof on thevirulence of a virus can also be determined using in vivo assays inwhich the titer of the virus in an infected subject administered anactive compound or composition thereof, the length of survival of aninfected subject administered an active compound or composition thereof,the immune response in an infected subject administered an activecompound or composition thereof, the number, duration and/or severity ofthe symptoms in an infected subject administered an active compound orcomposition thereof, and/or the time period before onset of one or moresymptoms in an infected subject administered an active compound orcomposition thereof, is assessed. Techniques known to one of skill inthe art can be used to measure such effects. In certain embodiments, anactive compound or composition thereof results in a 0.5 fold, 1 fold, 2fold, 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25 fold, 50fold, 75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200 fold, 300fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greater reductionin titer of influenza virus relative to an untreated subject. In someembodiments, an active compound or composition thereof results in areduction in titer of influenza virus relative to an untreated subjectof approximately 1 log or more, approximately 2 logs or more,approximately 3 logs or more, approximately 4 logs or more,approximately 5 logs or more, approximately 6 logs or more,approximately 7 logs or more, approximately 8 logs or more,approximately 9 logs or more, approximately 10 logs or more, 1 to 3logs, 1 to 5 logs, 1 to 8 logs, 1 to 9 logs, 2 to 10 logs, 2 to 5 logs,2 to 7 logs, 2 logs to 8 logs, 2 to 9 logs, 2 to 10 logs 3 to 5 logs, 3to 7 logs, 3 to 8 logs, 3 to 9 logs, 4 to 6 logs, 4 to 8 logs, 4 to 9logs, 5 to 6 logs, 5 to 7 logs, 5 to 8 logs, 5 to 9 logs, 6 to 7 logs, 6to 8 logs, 6 to 9 logs, 7 to 8 logs, 7 to 9 logs, or 8 to 9 logs.

Influenza virus animal models, such as ferret, mouse, guinea pig,squirrel monkey, macaque, and chicken, developed for use to testantiviral agents against influenza virus have been described. See, e.g.,Sidwell et al., Antiviral Res., 2000, 48:1-16; Lowen A. C. et al. PNAS.,2006, 103: 9988-92; and McCauley et al., Antiviral Res., 1995,27:179-186 and Rimmelzwann et al., Avian Diseases, 2003, 47:931-933. Formouse models of influenza, non-limiting examples of parameters that canbe used to assay antiviral activity of active compounds administered tothe influenza-infected mice include pneumonia-associated death, serumα1-acid glycoprotein increase, animal weight, lung virus assayed byhemagglutinin, lung virus assayed by plaque assays, andhistopathological change in the lung. Statistical analysis is carriedout to calculate significance (e.g., a P value of 0.05 or less).

In other assays, histopathologic evaluations are performed afterinfection of an animal model subject. Nasal turbinates and trachea maybe examined for epithelial changes and subepithelial inflammation. Thelungs may be examined for bronchiolar epithelial changes andperibronchiolar inflammation in large, medium, and small or terminalbronchioles. The alveoli are also evaluated for inflammatory changes.The medium bronchioles are graded on a scale of 0 to 3+ as follows: 0(normal: lined by medium to tall columnar epithelial cells with ciliatedapical borders and basal pseudostratified nuclei; minimal inflammation);1+ (epithelial layer columnar and even in outline with only slightlyincreased proliferation; cilia still visible on many cells); 2+(prominent changes in the epithelial layer ranging from attenuation tomarked proliferation; cells disorganized and layer outline irregular atthe luminal border); 3+ (epithelial layer markedly disrupted anddisorganized with necrotic cells visible in the lumen; some bronchiolesattenuated and others in marked reactive proliferation).

The trachea is graded on a scale of 0 to 2.5+ as follows: 0 (normal:Lined by medium to tall columnar epithelial cells with ciliated apicalborder, nuclei basal and pseudostratified. Cytoplasm evident betweenapical border and nucleus. Occasional small focus with squamous cells);1+ (focal squamous metaplasia of the epithelial layer); 2+ (diffusesquamous metaplasia of much of the epithelial layer, cilia may beevident focally); 2.5+ (diffuse squamous metaplasia with very few ciliaevident).

Virus immunohistochemistry is performed using a viral-specificmonoclonal antibody (e.g. NP-, N- or HN-specific monoclonal antibodies).Staining is graded 0 to 3+ as follows: 0 (no infected cells); 0.5+ (fewinfected cells); 1+ (few infected cells, as widely separated individualcells); 1.5+ (few infected cells, as widely separated singles and insmall clusters); 2+ (moderate numbers of infected cells, usuallyaffecting clusters of adjacent cells in portions of the epithelial layerlining bronchioles, or in small sublobular foci in alveoli); 3+(numerous infected cells, affecting most of the epithelial layer inbronchioles, or widespread in large sublobular foci in alveoli).

In one example, the ability to induce lung lesions and cause infectionin an animal model of virus infection is compared using wild-type virusand mock virus. Lung lesions can be assessed as a percentage of lunglobes that are healthy by visual inspection. Animals are euthanized 5days p.i. by intravenous administration of pentobarbital, and theirlungs are removed in toto. The percentage of the surface of eachpulmonary lobe that is affected by macroscopic lesions is estimatedvisually. The percentages are averaged to obtain a mean value for the 7pulmonary lobes of each animal. In other assays, nasal swabs can betested to determine virus burden or titer. Nasal swabs can be takenduring necropsy to determine viral burden post-infection.

In one embodiment, virus is quantified in tissue samples. For example,tissue samples are homogenized in phosphate-buffered saline (PBS), anddilutions of clarified homogenates adsorbed for 1 h at 37° C. ontomonolayers of cells (e.g., MDCK cells). Infected monolayers are thenoverlaid with a solution of minimal essential medium containing 0.1%bovine serum albumin (BSA), 0.01% DEAE-dextran, 0.1% NaHCO₃, and 1%agar. Plates are incubated 2 to 3 days until plaques could bevisualized. Tissue culture infectious dose (TCID) assays to titratevirus from PR8-infected samples are carried out as follows. Confluentmonolayers of cells (e.g., MDCK cells) in 96-well plates are incubatedwith log dilutions of clarified tissue homogenates in media. Two tothree days after inoculation, 0.05-ml aliquots from each well areassessed for viral growth by hemagglutination assay (HA assay).

5.13.6.1.1 Assays in Humans

In one embodiment, an active compound (e.g., chimeric influenzahemagglutinin (HA) polypeptides described herein) or composition thereofthat modulates replication of an influenza virus are assessed ininfected human subjects. In accordance with this embodiment, an activecompound or composition thereof is administered to the human subject,and the effect of the active compound or composition on viralreplication is determined by, e.g., analyzing the level of the virus orviral nucleic acids in a biological sample (e.g., serum or plasma). Anactive compound or composition thereof that alters virus replication canbe identified by comparing the level of virus replication in a subjector group of subjects treated with a control to that in a subject orgroup of subjects treated with an active compound or compositionthereof. Alternatively, alterations in viral replication can beidentified by comparing the level of the virus replication in a subjector group of subjects before and after the administration of an activecompound or composition thereof. Techniques known to those of skill inthe art can be used to obtain the biological sample and analyze the mRNAor protein expression.

In another embodiment, the effect of an active compound (e.g., chimericinfluenza hemagglutinin (HA) polypeptides described herein) orcomposition thereof on the severity of one or more symptoms associatedwith an influenza virus infection/disease are assessed in an infectedsubject. In accordance with this embodiment, an active compound orcomposition thereof or a control is administered to a human subjectsuffering from influenza virus infection and the effect of the activecompound or composition on one or more symptoms of the virus infectionis determined. An active compound or composition thereof that reducesone or more symptoms can be identified by comparing the subjects treatedwith a control to the subjects treated with the active compound orcomposition. In a specific embodiment, administration of an activecompound (e.g., chimeric influenza hemagglutinin (HA) polypeptidesdescribed herein) or composition thereof results in a decrease inhospitalization of a human or population of humans caused by influenzavirus disease or infection. In another specific embodiment,administration of an active compound or composition thereof results in areduced need for respiratory/breathing assistance in a human orpopulation of humans with an influenza virus disease or infection. Inanother specific embodiment, administration of an active compound orcomposition thereof results in a reduced length of illness of a human orpopulation of humans with an influenza virus disease or infection. Inanother specific embodiment, administration of an active compound orcomposition thereof results in improvement (e.g., an increase) in lungvolume as assessed by, e.g., whole body or lung plethysmography. Inanother embodiment, an active compound or composition thereof isadministered to a healthy human subject and monitored for efficacy as avaccine (e.g., the subject is monitored for the onset of symptoms ofinfluenza virus infection; the ability of influenza virus to infect thesubject; and/or a reduction in/absence of one or more symptomsassociated with influenza virus infection). Techniques known tophysicians familiar with infectious diseases can be used to determinewhether an active compound or composition thereof reduces one or moresymptoms associated with the influenza virus disease.

5.14 Assessment of Antibodies in a Subject

In another aspect, a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein, or virus expressing a chimeric influenza hemagglutinin(HA) polypeptide described herein, can be used to assess the antibodyresponse of a subject (e.g., a naive subject or an immunized/vaccinatedsubject) or a population of subjects to an influenza virus hemagglutininpolypeptide (e.g., a chimeric influenza hemagglutinin (HA) polypeptidedescribed herein). In specific embodiments, a chimeric influenza virushemagglutinin polypeptide or a virus expressing a chimeric influenzavirus hemagglutinin polypeptide can be used to assess the presence ofstem-specific antibodies in the subject or population of subjects.

In a specific embodiment, the antibody response of a subject or apopulation of subjects that has been an immunized/vaccinated with aninfluenza virus hemagglutinin polypeptide (e.g., a chimeric influenzahemagglutinin (HA) polypeptide described herein, or a virus expressing achimeric influenza hemagglutinin (HA) polypeptide described herein), isassessed to identify the types of stalk-specific antibodies in thesubject or population of subjects. Such an assessment may allow for theidentification surrogate markers/endpoints important in determining theclinical response to administration of an influenza virus HA polypeptidepolypeptide(s) (e.g., a chimeric influenza hemagglutinin (HA)polypeptide described herein, or a virus expressing a chimeric influenzahemagglutinin (HA) polypeptide described herein) described herein. Insuch an approach, a biological sample, e.g., blood, from the subject orpopulation of subjects may be isolated and tested directly for thepresence of antibodies, or may be processed (e.g., to obtain sera) andsubsequently tested for the presence of antibodies. Such antibodytesting can utilize assays known in the art, e.g., ELISA.

In another specific embodiment, the antibody profile of a naive subject(i.e., a subject that has not been immunized/vaccinated with aninfluenza virus HA polypeptide polypeptide(s) (e.g., a chimericinfluenza hemagglutinin (HA) polypeptide described herein), or a virusexpressing an influenza virus HA polypeptide polypeptide(s) (e.g., achimeric influenza hemagglutinin (HA) polypeptide described herein)) ora population of naive subjects is assessed to determine whether saidsubject or population of subjects possesses globular head-specificand/or stem specific antibodies against various influenza virus strainsor subtypes. Such an assessment may allow for the generation of achimeric influenza hemagglutinin (HA) polypeptide, or viruses expressingchimeric influenza hemagglutinin (HA) polypeptides, that are suitablefor administration to said subject or population of subjects. Such anassessment may determine an immunization strategy for the patient.

In another specific embodiment, provided herein is a method ofassessing/detecting the presence of antibodies in a subject that arespecific for a stem domain of a particular influenza virus strain orsubtype comprising contacting in vitro a biological sample (e.g., blood,sera) from said subject with a chimeric influenza virus hemagglutininpolypeptide described herein, wherein said chimeric influenza virushemagglutinin polypeptide comprises a stem domain from the strain orsubtype of interest. In another specific embodiment, provided herein isa method of assessing/detecting the presence of antibodies in a subjectthat are specific for a stem domain of a particular influenza virusstrain or subtype comprising contacting in vitro a biological sample(e.g., blood, sera) from said subject with a virus expressing/containinga chimeric influenza virus hemagglutinin polypeptide described herein,wherein said chimeric influenza virus hemagglutinin polypeptidecomprises a stem domain from the strain or subtype of interest.

5.15 Kits

Provided herein is a pharmaceutical pack or kit comprising one or morecontainers filled with one or more of the ingredients of thepharmaceutical/immunogenic compositions described herein, such as one ormore active compounds provided herein. Optionally associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

The kits encompassed herein can be used in accordance with the methodsdescribed herein. In one embodiment, a kit comprises an active compounddescribed herein, preferably one or more chimeric influenzahemagglutinin (HA) polypeptide described herein, in one or morecontainers. In certain embodiments, a kit comprises a vaccine describedherein, e.g., a split virus vaccine, a subunit vaccine, an inactivatedinfluenza virus vaccine, or a live influenza virus vaccine, wherein saidvaccine comprises one or more chimeric influenza hemagglutinin (HA)polypeptides described herein. In a specific embodiment, provided hereinare kits comprising a chimeric influenza virus hemagglutinin polypeptidedescribed herein and instructions for using the chimeric influenza virushemagglutinin polypeptide to assess the antibodies present in a subject.In another specific embodiment, provided herein are kits comprising achimeric influenza virus hemagglutinin polypeptide described herein foruse in methods of assaying for the presence of HA stem domain specificantibodies in a sample.

In a specific embodiment, a kit provided herein comprises a cH5/1chimeric influenza hemagglutinin polypeptide described herein (or anucleic acid encoding such a polypeptide, a vector (e.g., a viralvector, or a bacteria) containing or expressing such a polypeptide, orcells stimulated with such a polypeptide), a cH5/3 chimeric influenzahemagglutinin polypeptide described herein (or a nucleic acid encodingsuch a polypeptide, a vector (e.g., a viral vector, or a bacteria)containing or expressing such a polypeptide, or cells stimulated withsuch a polypeptide), a cH7/3 chimeric influenza hemagglutininpolypeptide described herein (or a nucleic acid encoding such apolypeptide, a vector (e.g., a viral vector, or a bacteria) containingor expressing such a polypeptide, or cells stimulated with such apolypeptide), a cH5/B chimeric influenza hemagglutinin polypeptidedescribed herein (or a nucleic acid encoding such a polypeptide, avector (e.g., a viral vector, or a bacteria) containing or expressingsuch a polypeptide, or cells stimulated with such a polypeptide), acH7/B chimeric influenza hemagglutinin polypeptide described herein (ora nucleic acid encoding such a polypeptide, a vector (e.g., a viralvector, or a bacteria) containing or expressing such a polypeptide, orcells stimulated with such a polypeptide), or a cHB/B chimeric influenzahemagglutinin polypeptide described herein (or a nucleic acid encodingsuch a polypeptide, a vector (e.g., a viral vector, or a bacteria)containing or expressing such a polypeptide, or cells stimulated withsuch a polypeptide).

In another specific embodiment, a kit provided herein comprises acombination of a cH5/1 chimeric influenza hemagglutinin polypeptidedescribed herein (or a nucleic acid encoding such a polypeptide, avector (e.g., a viral vector, or a bacteria) containing or expressingsuch a polypeptide, or cells stimulated with such a polypeptide) and acH5/3 chimeric influenza hemagglutinin polypeptide described herein (ora nucleic acid encoding such a polypeptide, a vector (e.g., a viralvector, or a bacteria) containing or expressing such a polypeptide, orcells stimulated with such a polypeptide); or a combination of a cH5/1chimeric influenza hemagglutinin polypeptide described herein (or anucleic acid encoding such a polypeptide, a vector (e.g., a viralvector, or a bacteria) containing or expressing such a polypeptide, orcells stimulated with such a polypeptide) and a cH7/3 chimeric influenzahemagglutinin polypeptide described herein (or a nucleic acid encodingsuch a polypeptide, a vector (e.g., a viral vector, or a bacteria)containing or expressing such a polypeptide, or cells stimulated withsuch a polypeptide).

In another specific embodiment, a kit provided herein comprises acombination of a cH5/1 chimeric influenza hemagglutinin polypeptidedescribed herein (or a nucleic acid encoding such a polypeptide, avector (e.g., a viral vector, or a bacteria) containing or expressingsuch a polypeptide, or cells stimulated with such a polypeptide) and acH5/3 chimeric influenza hemagglutinin polypeptide described herein (ora nucleic acid encoding such a polypeptide, a vector (e.g., a viralvector, or a bacteria) containing or expressing such a polypeptide, orcells stimulated with such a polypeptide) and either of a cH5/B, acH7/B, or a cB/B chimeric influenza hemagglutinin polypeptide describedherein (or a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, or cells stimulated with such a polypeptide).

In another specific embodiment, a kit provided herein comprises acombination of a cH5/1 chimeric influenza hemagglutinin polypeptidedescribed herein (or a nucleic acid encoding such a polypeptide, avector (e.g., a viral vector, or a bacteria) containing or expressingsuch a polypeptide, or cells stimulated with such a polypeptide) and acH7/3 chimeric influenza hemagglutinin polypeptide described herein (ora nucleic acid encoding such a polypeptide, a vector (e.g., a viralvector, or a bacteria) containing or expressing such a polypeptide, orcells stimulated with such a polypeptide) and either of a cH5/B, acH7/B, or a cB/B chimeric influenza hemagglutinin polypeptide describedherein (or a nucleic acid encoding such a polypeptide, a vector (e.g., aviral vector, or a bacteria) containing or expressing such apolypeptide, or cells stimulated with such a polypeptide).

6. Examples 6.1 Example 1 Chimeric Influenza Virus HemagglutininPolypeptides

This example describes chimeric influenza virus hemagglutininpolypeptides and methods for inducing high levels of cross-neutralizingHA stalk antibodies in a subject comprising administration of saidchimeric influenza virus hemagglutinin polypeptides. As described inthis example, chimeric influenza virus hemagglutinin were generated thatwere successfully expressed by influenza virus and by cells engineeredto express the chimeric influenza virus hemagglutinin. The chimericinfluenza virus hemagglutinin were successfully recovered in theirproper conformation, as evidenced by antibody recognition of both thestem and head domains of the chimeric influenza virus hemagglutinin.

FIG. 2 depicts chimeric influenza virus hemagglutinins (HAs), comprisingthe stem/stalk domain of an H1 subtype of influenza virus and theheterologous globular head domains of other influenza virus subtypes(H2, H3, and H5). Following the strategy outline in FIG. 2, an influenzavirus was generated that comprises a chimeric HA composed of a stemdomain derived from an H1N1 (PR8-H1N1) influenza virus and the globularhead domain of the 2009 pandemic H1 HA (Cal/09). The globular headdomains of the HAs of the two viruses are very distinct (˜70% amino acididentity) whereas the stem domains are highly conserved but stilldivergent (˜89% amino acid identity). As demonstrated in FIG. 3, thechimeric HAs with the same stem domain but very different HA headswithin the same subtype were expressed.

In addition, a chimeric HA consisting of the stalk domain of A/PR8/34 HAand the globular head domain of HK/68 (chimeric H3) as well as wild typeHAs (PR8-HA and HK68 HA) were expressed in 293T cells. FIG. 5demonstrates that it is also possible to express stable chimeric HAswith the same stem domain (derived from the H1 subtype HA) and with aglobular head from a different subtype (H3).

Thus, HA immunogens that completely share the HA stem domain but arehighly divergent in their globular heads were designed. Repeatedimmunizations with these constructs should result in high levels ofcross-neutralizing antibodies against the common stem domain of the HA.An improved vaccine strategy thus uses chimeric HAs with a constantstem/stalk domain and a changing globular head to induce robustcross-neutralizing anti-stem domain antibodies. A constant stem domainof, e.g., the H1 HA from A/PR/8/34 can be used together with globularheads from different group 1 HAs (H1, H2, H5, H9) to make a panel ofeither recombinant inactivated viruses, recombinant attenuated virusesor recombinant HAs (FIG. 4). A similar panel for group 2 HAs based onthe stem domain of, e.g., H3 HA of an X31 virus, in combination with H3,H4 and H7 globular heads can provide the basis for a group 2 HAuniversal vaccine. Recombinant viruses can be rescued on an influenzavirus vaccine backbone, such as PR/8 or cold-adapted influenza viruses,grown by standard techniques and used as inactivated or attenuatedvaccines. Recombinant HAs can be expressed in insect cells that are ableto perform mammalian-like glycosylation (MIMIC Sf9) or by transienttransfection of, e.g., 293 T or Vero cells, and then can be purified byNi-chelat chromatography with the help of a C-terminal his tag. Otherstrategies can include the use of DNA vaccines expressing the chimericHAs or other vectors, such as adenovirus vectors, expressing thechimeric HAs.

6.2 Example 2 Viruses Expressing Chimeric Influenza Virus HemagglutininPolypeptides

This example describes several functional chimeric influenza virushemagglutinins encompassing a variety of globular head and stalkcombinations from different hemagglutinin subtypes as well asrecombinant influenza viruses expressing these chimeric hemagglutinins,which had growth properties similar to those of wild-type influenzaviruses.

6.2.1 Materials and Methods 6.2.1.1 Cells and Viruses

293T and MDCK cells were obtained from the American Type CultureCollection (ATCC, Manassas, Va.) and were maintained either inDulbecco's minimal essential medium (DMEM) or in MEM (Gibco, Invitrogen)supplemented with 10% fetal calf serum (HyClone; Thermo Scientific) andpenicillin-streptomycin (Gibco, Invitrogen).

All A/PR/8/34 recombinant viruses were grown in 10-day old embryonatedchicken eggs at 37° C. for 2 days.

6.2.1.2 Construction of Plasmids

Plasmids encoding the different chimeric hemagglutinins were constructedby a similar strategy adapted from constructing reverse geneticsplasmids for generating recombinant viruses as previously described(see, e.g., Fodor et al., 1999, J Virol 73:9679-9682; and Hai et al.,2008, J Virol 82:10580-10590). Briefly, the different segments ofchimeric HA were amplified by PCR with primers containing SapI sites,digested with SapI, and cloned into the SapI sites of the pDZ vectorthat contains the human RNA polymerase I promoter and the mouse RNApolymerase I terminator (see, e.g. Quinlivan et al., 2005, J Virol79:8431-8439), through multi-segmental ligation.

6.2.1.3 Flow Cytometric Analysis

To assess levels of hemagglutinin proteins at the cell surface, 293Tcells were transfected with 1 μg of the appropriate plasmid usingLipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions. At 24 h post-transfection, cells were trypsinized andresuspended in PBS containing 2% FBS prior to staining them with themonoclonal antibody (mAb) 6F12 against H1 HAs at a 1/1000 dilution orwith the mAb 12D1 against H3 HAs (see Wang et al., 2010, PLoS Pathog6:e1000796) at a 1/400 dilution. Stained cells were enumerated on aBeckman Coulter Cytomics FC 500 flow cytometer, and the results wereanalyzed using FlowJo software.

6.2.1.4 Pseudoparticle Generation and Entry Assay

The procedure for pseudo-particle production was adapted from previousstudies (see, e.g., Evans et al., 2007, Nature 446:801-805; and Sui etal., 2011, Clin Infect Dis 52:1003-1009). Briefly, 293-T cells wereco-transfected with four plasmids encoding (i) a pro-virus containingthe desired reporter (V1-GLuc), (ii) HIV Gag-Pol, (iii) the differentchimeric hemagglutinin protein and (iv) influenza A PR8 neuraminidase(NA). Supernatants were collected 72 h post-transfection andsubsequently, filtered (0.45 μm pore size). All transductions andinfection assays using pseudo-particles were performed in the presenceof 1 μg/ml polybrene (Sigma, St. Louis, Mo.) (see Sui et al., 2011, ClinInfect Dis 52:1003-1009).

The entry assay was performed through infecting MDCK cells withpseudo-particles with different chimeric hemagglutinin containing theG-Luc reporter. Twenty-four hours post-infection, cells were washedthree times with fresh medium to remove G-Luc protein that was presentin the pseudo-particle inoculum. Forty-eight hours post-infectionluciferase assays were performed (see Evans et al., 2007, Nature446:801-805).

6.2.1.5 Rescue of Recombinant Chimeric Influenza a Viruses

Rescue of influenza A viruses from plasmid DNA was performed aspreviously described (see, e.g., Fodor et al., 1999, J Virol73:9679-9682; and Hai et al., 2008, J Virol 82:10580-10590). To generatethe recombinant wild-type (rWT) PR8 virus, 293T cells wereco-transfected with 1 μg of each of the 8 pDZ PR8 rescue plasmids usingLipofectamine 2000 (Invitrogen, Carlsbad, Calif.). The virusesexpressing different chimeric HA were generated in the same way butsubstituting the HA plasmid by the corresponding chimeric one to recoverthe corresponding chimeric viruses. At 6 h post-transfection, the mediumwas replaced with DMEM containing 0.3% bovine serum albumin (BSA), 10 mMHEPES, and 1.5 μg/ml TPCK (L-1-tosylamide-2-phenylethyl chloromethylketone)-treated trypsin. After 24 hours post-transfection,virus-containing supernatant was inoculated into 8-day old embryonatedchicken eggs. Allantoic fluid was harvested after 2 days of incubationat 37° C. and assayed for the presence of virus by hemagglutination ofchicken red blood cells and by plaque formation in MDCK cells.

6.2.1.6 Virus Growth Kinetics Assay

To analyze the replication characteristics of recombinant viruses,10-day old embryonated chicken eggs were inoculated with 100 pfu of eachrespective virus. Allantoic fluid was harvested and subsequently assayedfor viral growth at 0, 9, 24, 48, and 72 h post-infection (hpi). Thetiters of virus present in allantoic fluid were determined by plaqueassay on MDCK cells.

6.2.1.7 Immunostaining of Plaques

Plaques were visualized by immunostaining with the mAb (HT103) againstthe influenza A NP protein.

6.2.1.8 Western Blot and Indirect Immunofluorescence Analysis

One well of a 12-well dish of confluent MDCK cells was infected(multiplicity of infection [MOI] of 2) with indicated recombinantinfluenza viruses or mock infected with phosphate-buffered saline (PBS)for 1 h at 37° C. At 12 h post-infection (hpi), cells were lysed in lxprotein loading buffer as described previously (see, e.g., Hai et al.,2008, J Virol 82:10580-10590). The reduced cell lysates were analyzed byWestern blot analysis by using mAbs against, A/NP (HT103), A/PR8/HA(PY102), A/Cal/09/HA (29C1), A/VN/HA (M08) (20), A/H3/HA (12D1). Thedetection of Perth-cH7 used a goat polyclonal sera, NR-3152, againstA/FPV/Dutch/27 (H7) virus, which was obtained from the BEI Resources.The mAb anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loadingcontrol antibody was from Abcam. All the proteins of interest werevisualized using an enhanced chemiluminescence protein detection system(PerkinElmer Life Sciences, Boston, Mass.).

For immunofluorescence analysis, confluent monolayers of MDCK cells on15-mm coverslips were infected with recombinant viruses at an MOI of 2.At 15 hpi, cells were fixed and permeabilized with methanol-acetone(ratio, 1:1) at −20° C. for 20 min. After being blocked with 1% bovineserum albumin in PBS containing 0.1% Tween 20, cells were incubated for1 h with the antibody directed against A/NP (HT103), A/H1/HA (6F12),A/PR8/HA (PY102), A/Cal/09/HA (29C1), A/VN/HA (M08) (20), A/H3/HA(12D1), and A/H7 virus (NR-3152) as mentioned above. After three washeswith PBS containing 0.1% Tween 20, cells were incubated for 1 h withAlexa Fluor 594-conjugated anti-mouse immunoglobulin G (IgG; Invitrogen,Carlsbad, Calif.) or Alexa Fluor 594-conjugated anti-goat immunoglobulinG (IgG, Invitrogen, Carlsbad, Calif.). Following the final three washes,infected cells were analyzed by fluorescence microscopy with an OlympusIX70 microscope.

6.2.2 Results 6.2.2.1 Generation of Chimeric Hemagglutinins

In order to gain information regarding the conservation of the cysteineresidues forming the Cys52-Cys277 disulfide bond, an alignment of theinfluenza A virus HA sequences of the H1, H3, H5 and H7 subtypes wasgenerated (see FIG. 6). The sequences of the HA1 subunits are lessconserved than those of the HA2 subunits, mainly due to the presence ofimmuno-dominant antigenic sites on the globular head domain. The moreconserved HA2 chains comprise the stalk regions anchoring the HAmolecules to the viral envelope. The alignment demonstrates that Cys52and Cys277 and the amino acids towards both ends are conserved acrossselected subtypes. Henceforth, the hemagglutinin sequences N-terminal toCys52 and C-terminal to Cys277 are defined as the stalk domain (FIG. 6).The intervening sequence is considered in this example to be the headdomain.

A chimeric hemagglutinin construct (PR8-cH1) containing the pandemic H1Cal/09 HA globular head domain with the stalk region from the PR8 (H1)HA was first generated (FIG. 7A). A chimeric HA (PR8-cH5) containing theglobular head from the VN1203 (H5) HA with the stalk from the PR8 (H1)HA also was generated (FIG. 7B). Since all 16 subtypes of influenza HAare grouped into two phylogenetic groups (groups 1 and 2) (see, e.g.,Sui et al., 2009, Nat Struct Mol Biol 16:265-273) and H1 and H5 HAs bothbelong to group 1, a similar strategy to generate a chimeric HA bearingthe A/Alberta/24/01 (H7) head domain with the stalk region fromA/Perth/16/2009 (H3) HA (Perth-cH7) (FIG. 7B) was applied. Both H7 andH3 are members from the group 2 phylogenetic group (see, e.g., Sui etal., 2009, Nat Struct Mol Biol 16:265-273).

It was next tested whether the different chimeric HA constructs werebeing expressed and transported to the cell surface.Fluorescence-activated cell sorter (FACS) analysis of transientlytransfected 293T cells was performed following surface staining with PR8and H3 stalk domain specific antibodies, respectively (FIG. 8A). Asshown in FIG. 8A, expression of all three chimeric constructs wasdetected. This detection indicates that the transportation of thechimeric HAs through the Golgi complex to the cell surface is notdisrupted.

Next the entry characteristics of the different chimeric HAs wasexamined through infection of MDCK cells with retroviral HA-pseudotypedparticles containing the chimeric HA, wild type influenza A PR8 NA andHIV-based luciferase. The entry efficiency mediated by the chimeric HAproteins was detected by luciferase read-out. Comparable levels ofpseudotyped particle-mediated luciferase delivery were observed forPR8-cH5 and Perth-cH7 chimeric HAs and the corresponding wild typeproteins (FIG. 8B). The PR8-cH1 HA showed a lower luciferase levelcompared to the other HA constructs.

6.2.3 Generation of Recombinant Influenza Viruses Bearing ChimericHemagglutinins.

In light of the above results, whether a chimeric HA is functional inthe context of a whole virus particle and ultimately would allow therescue of a recombinant influenza A virus only expressing a chimeric HAwas ascertained. Using previously published protocols (see, e.g., Fodoret al., 1999, J Virol 73:9679-9682; and Hai et al., 2008, J Virol82:10580-10590), viruses containing all of the different chimeric HAswere successfully generated. The resulting viruses were plaque purified,amplified in 10-Day-old embryonic eggs and the chimeric segments wereanalyzed by RT-PCR and sequenced. In all cases, the virus was found tohave the expected chimeric HA segment and no other HA segments.

The identity of the chimeric viruses was further demonstrated by Westernblotting and indirect immunofluorescence of infected cells (FIGS. 9A and9B). MDCK cells were infected with rWT A/PR8, wild-type A/Perth,PR8-cH1, PR8-cH5, and Perth-cH7 viruses (FIGS. 9A and 9B). PR8-cH1 andPR8-cH5 chimeric HA proteins were detected in the corresponding samplesusing antibodies against either Cal/09 HA (29C1) or VN/04 HA (M08) (seeSteel et al., 2009, J Virol 83:1742-1753), respectively (FIG. 9A). Using12D1, a pan H3 stalk mAb (see Wang et al., 2010, PLoS Pathog6:e1000796), comparable expression levels between the Perth cH7-HA andwild type Perth HA were observed. A positive band was only detected forthe Perth-cH7 infection sample when using anti-H7 antibodies (NR-3152).

For the immunofluorescence study, the infection conditions were similarto those for the Western analysis. Infected cells were stained withcorresponding antibodies as indicated in FIG. 9B. All the infected cellsshowed the expected expression of the HAs as well as of the A/NP protein(FIG. 9B).

6.2.4 Replication Characteristics of Recombinant Viruses

The growth properties of the viruses were assessed in 10-day-oldembryonated chicken eggs at 37° C. (FIG. 8A). The rWT PR8 virus wasincluded for comparison of the growth kinetics of the recombinantviruses expressing chimeric HAs. Regarding the PR8-cH5 virus, a similarreplication pattern as compared to PR8 virus was observed. As forPerth-cH7 virus, there was a 2 fold reduction in viral titer compared tothe rWT PR8 virus at 9 hpi. Nevertheless, it reached a similar peaktiter as the wild type virus (1×109 PFU/ml) at 48 hpi. The PR8-cH1 viruswas attenuated when compared to the rWT PR8 virus, as shown by reductionin titers through all the time points. Nonetheless, even this chimericvirus reached a respectable peak titer of approximately 108 PFU/ml. Theplaque phenotype of each of the chimeric viruses was also evaluated inMDCK cells. All viruses formed comparably sized plaques as shown in FIG.8B. These results confirm that the chimeric HA constructs fold correctlyin vivo and are biologically functional.

6.2.5 Conclusion

A novel strategy was developed to generate influenza viruses withchimeric HA proteins bearing different HA globular head domains bytaking advantage of the conserved disulfide bond Cys52-Cys277 whichdemarcates the border between the head and stalk domains. Thus, throughsubstituting the parental head domain with the head domain of anotherHA, a panel of chimeric HAs with the same stalk but different globularheads was generated. The design was tested across multiple subtypes,including the PR8 stalk domain with Cal/09 and VN H5 globular heads. Inaddition, an H7 globular head was placed on an H3 stalk domain. Theseconstructs cover both phylogenetic groups of the influenza HA protein.Each construct was expressed on the cell surface and retained fusionactivity as shown in FIG. 7. The generation of recombinant virusesbearing the chimeric HAs further validated that the HAs fold correctlyand retain biological functions.

6.3 Example 3 Diagnostic Applications of Chimeric Influenza VirusHemagglutinin Polypeptides and Vaccination of Mice with ChimericInfluenza Virus Hemagglutinin Polypeptides

This example demonstrates that chimeric influenza virus hemagglutininpolypeptides can be utilized for diagnostic purposes and that virusesexpressing chimeric influenza virus hemagglutinin polypeptides can beutilized in vaccines.

6.3.1 Materials and Methods 6.3.1.1 Cells and Plasmids

293T and MDCK cells were obtained from ATCC and were maintained inDulbeccos's Modified Eagle's medium (DMEM) and Minimal Essential Medium(both from Gibco), respectively, each supplemented with 10% fetal calfserum (HyClone), and 100 units/ml of penicillin-100 μg/ml ofstreptomycin (Pen/Strep, Gibco). TNM-FH (Sigma-Aldrich) supplementedwith 10% fetal calf serum and Hyclone SFX insect culture media(ThermoScientific) were used for Sf9 and BT1-TN5B1-4 (High Five) cellculture.

Chimeric hemagglutinin constructs with the stalk of A/Puerto Rico/8/1934(PR8) containing the globular head domain from eitherA/Mallard/Sweden/81/02 (“cH6”) virus or A/Guinea fowl/Hong Kong/WF10/99(“cH9”) virus were generated using similar techniques. For the chimericH6 construct, different components were amplified by PCR and cloned intothe pDZ plasmid using a cloning strategy previously described (see,e.g., Fodor et al., 1999, J Virol 73:9679-82; and Hai et al., 2008,Journal of Virology 82:10580-90). Briefly, different components of thechimeric hemagglutinin (cHA) were amplified by PCR with primerscontaining Sap I sites, digested with Sap I, and cloned into the Sap Isites of the pDZ plasmid. For generation of the baculo-transfer plasmid,cH6 was amplified by PCR, cut with BamHI and NotI, and cloned in frameinto a modified pBacPAK8 (Gentech) baculo-transfer vector that harbors aC-terminal T4 phage foldon and a 6-his tag (see Meier et al., 2004, JMol Biol 344:1051-69). The sequences of all plasmids were confirmed bySanger sequencing.

6.3.1.2 Rescue of Recombinant cH9 Virus

In order to rescue the recombinant vaccine virus, eight reverse geneticsplasmids that encode vRNA and mRNA of the seven wild type viral segmentsfrom PR8 and the cH9 were used, as previously described (Fodor et al.,1999, J Virol 73:9679-82; and Pleschka et al., 1996, J Virol70:4188-92). Briefly, 293T cells were transfected with 1 μg of each ofthe eight plasmids using Lipofectamine 2000 (Invitrogen). At 12 hourspost transfection, the medium was replaced with DMEM containing 0.3%bovine serum albumin, 10 mM HEPES, and 1.5 μg of TPCK(l-1-tosylamide-2-phenylethyl chloromethyl ketone)-treated trypsin/mL.At two days post transfection, virus-containing supernatant wasinoculated into 10-day-old embryonated chicken eggs. Allantoic fluid washarvested after 2 days of incubation at 37° C. and assayed for thepresence of virus by the hemagglutination of chicken red blood cells.The rescued cH9 virus was then propagated in 10-day old eggs following a48 hour incubation at 37° C. Virus stocks were tittered by plaque assayas previously described (see, e.g., Steel et al., 2009, Journal ofVirology 83:1742-53). The sequence of the cH9 was confirmed bysequencing of reverse transcription-PCR products.

6.3.1.3 Recombinant Baculovirus Generation, Protein Expression andPurification

In order to generate recombinant baculoviruses (rBV) carrying the cH6,plasmids and Baculogold DNA (BDBioschiences) were co-transfected intoSf9 cells with Cellfectin II (Invitrogen) according to themanufacturer's instructions. Recombinant baculovirus was amplified inSf9 cells grown in TNM-FH medium (Gemini Bioproducts, West Sacramento,Calif.) and titers were determined by plaque assay as previouslydescribed (see, e.g., Steel et al., 2009, Journal of Virology83:1742-53).

High Five cells (see Krammer et al., 2010, Mol Biotechnol 45:226-34)grown in HyClone SFX insect cell media (Thermo Fisher Scientific,Waltham, Mass.) were infected with rBV expressing cH6 at a multiplicityof infection (MOI) of 10 and a cell density of 1×10⁶ cells/ml in 500 mlshaker flasks. Cells were harvested 96 hours post-infection andseparated from supernatant by low speed centrifugation for 10 minutes at2000×g at room temperature. For purification of cH6 protein, thesupernatant was collected and incubated with Ni-NTA resin (Qiagen) for 2hours at 4° C. The slurry was loaded on columns and washed trice withwashing buffer (50 mM Na2HCO3, 300 mMNaCl, 20 mM Imidazole, pH 8).Protein was eluted in 0.5 ml steps with elution buffer (50 mM Na2HCO3,300 mMNaCl, 250 mM Imidazole, pH 8), tested for protein content withBradford reagent and fractions containing protein were pooled. Proteinpurity and identity was tested by SDS-PAGE, Coomassie staining andWestern blot. Protein concentration was determined with Bradfordreagent.

6.3.1.4 Mouse Experiments

For all procedures, mice were anesthetized with intraperitonealinjections of 0.1 mL of ketamine/xylazine mixtures (1.5 mg ketamine and0.3 mg xylazine).

Mouse 50% lethal doses (LD₅₀) were determined in BALB/c mice (CharlesRiver Laboratories) by infecting groups of four mice with 10-fold serialdilutions of influenza virus. Body weights were monitored for a two weekperiod. Mice that lost greater than 25% of their body weight wereconsidered to have reached experimental endpoint and were euthanized.LD₅₀ values were calculated by the method of Reed and Meunch (see Reedand Meunch. 1938, Am. J. Hyg. 27:493-497).

For experiments to assess the stem antibodies produced followingA/California/04/09 (Cal/09) virus infection, female, 8-10 week oldBALB/c mice were first primed with intramuscular administration of 80 μgof an expression plasmid encoding full length HA from PR8 virus, coupledwith the application of electrical stimulation as previously described(TriGrid delivery system, Ichor Medical Systems) (see, e.g., Luxembourget al., 2007, Expert Opinion on Biological Therapy 7:1647-64). Threeweeks later, mice were boosted with a sublethal dose of 10⁴ PFU Cal/09in a 50 μl volume, intranasally. Control animals either received DNAalone or Cal/09 virus alone, or intramuscular injection of the 2009-2010vaccine (Cal/09 split vaccine). Three weeks post boost, or theequivalent time point for control animals, mice were bled and sera washarvested to test reactivity to cH6 by immunofluorescence as describedbelow.

For experiments testing the efficacy of cH9 virus as a vaccineconstruct, PR8 full length DNA was administered as described above.Three weeks post prime, mice were inoculated with 10³ PFU of cH9 virusinstilled intranasally. Control animals either received DNA alone orCal/09 virus alone or purified inactivated PR8 virus intramuscularly.Three weeks post boost, or the equivalent time point for controlanimals, mice were challenged with intranasal inoculation of 5×10⁴ LD₅₀of PR8 virus. Mice were weighed for 14 days post challenge. Animals thatlost more than 27.5% of their initial body weight were euthanized andscored as dead.

6.3.1.5 Immunofluorescence to Confirm Expression of ChimericHemagglutinin

Confluent monolayers of 293T cells were transfected with 1 μg of pDZ cH6plasmid. At 48 hours post transfection, cells were fixed and blockedwith 1% bovine serum albumin in PBS containing 0.1% Tween 20. Cells werethen incubated with sera pooled from the animals of each of the fourexperimental groups described above (PR8 DNA alone, Cal/09 alone, PR8DNA and Cal/09 infection, or Cal/09 split vaccine alone). After threewashes with PBS containing 0.1% Tween 20, cells were incubated for 1hour with Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen).Infected cells were analyzed by fluorescence microscopy with an OlympusIX70 microscope.

6.3.1.6 ELISA

Ninety-six well ELISA plates (Nunc, MaxiSorp) were coated with 50 ml ofbaculovirus-expressed cH6 and incubated overnight at 4° C. Plates wereblocked 3% milk/PBS and then washed with PBS/0.1% Tween (PBST). Serumfrom vaccinated mice was serially diluted in PBS and added to the plate,followed by a 1 hour incubation at 37° C. Plates were then washed withPBST and incubated with 1:2500 dilution of horseradish peroxidase linkedanti-mouse IgG (GE Healthcare). Following an additional wash with PBST,SigmafastOPD substrate (Sigma) was added. The reaction was stopped with3M H₂SO₄ and optical density measurements were taken at 490 nm.

6.3.2 Results 6.3.2.1 Chimeric Influenza Virus HemagglutininPolypeptides can be Used in Diagnostic Applications

A chimeric hemagglutinin construct comprising the globular head domainfrom the hemagglutinin of an H6 influenza virus subtype and stem/stalkdomain from the hemagglutinin of the PR8 virus was generated to serve asanalytical tool to assay production of antibodies by the immunized miceagainst the H6 stem domain. Because the immunized mice were only exposedto the globular head of H1 viruses, antibodies that were generated inthe experimental animals would only be reactive to chimeric H6hemagglutinin (cH6) if they were directed towards its H1 stem.

As shown in FIGS. 11A and 11D, treatment with DNA alone or pandemicsplit vaccine did not elicit any stem reactive antibodies in thevaccinated mice. Conversely, infection with Cal/09 alone generated stemreactive antibodies (FIG. 11B), though not to the extent elicited by DNAelectroporation and infection (FIG. 11C). A cross-reactive H1 stemantibody, C179, was used as a control for the transfection (FIG. 11E).As expected, PY102, an antibody directed against the globular head ofPR8, did not react to the transfected cH6 HA (FIG. 11F).

As shown in FIG. 13, the utility of the cH6 chimeric influenza virushemagglutinin polypeptide as a tool in which to detect stem antibodybinding was confirmed by ELISA.

6.3.2.2 Chimeric Influenza Virus Hemagglutinin Polypeptides can be Usedin Vaccines

As shown in FIG. 12, animals that were vaccinated with inactivated PR8virus were protected from lethal challenge, while animals that receivedDNA alone completely succumbed to infection by day 5 post challenge.Animals that received cH9 virus alone also were not protected frominfection, with only a 25% survival rate. By contrast, animals that werefirst primed with DNA and then boosted with cH9 virus were protectedfrom challenge, with a survival rate that was statistically the same asanimals vaccinated with the inactivated virus preparation.

6.4 Example 4 Hemagglutinin Stalk Antibodies Elicited by Infection withthe 2009 Pandemic H1N1 Influenza Virus

This example describes chimeric influenza virus hemagglutininpolypeptides that were used to study stem domain specific antibodies.Using these polypeptides, it was determined that infection with the 2009pandemic H1N1 virus elicited a boost in titer of virus-neutralizingantibodies directed against the hemagglutinin stem. In addition to thechimeric influenza virus hemagglutinin polypeptides, assays that can beused to measure influenza virus-neutralizing antibodies which are notdetected in the traditional hemagglutination-inhibition assay are alsodescribed.

6.4.1 Materials and Methods 6.4.1.1 Cells and Plasmids

293T and MDCK cells were obtained from ATCC and were maintained inDulbeccos's Modified Eagle's medium (DMEM) and Minimal Essential Medium(both from Gibco), respectively, each supplemented with 10% fetal calfserum (HyClone), and 100 units/ml of penicillin-100 μg/ml ofstreptomycin (Pen/Strep, Gibco). TNM-FH media (Gemini Bioproducts)supplemented with 10% fetal calf serum and Hyclone SFX insect culturemedia (ThermoScientific) were used for Sf9 and BT1-TN5B1-4 (High Five)cell culture.

Chimeric hemagglutinin (cHA) constructs with the stalk of A/PuertoRico/8/1934 (PR8) containing the globular head domain from eitherA/Mallard/Sweden/81/02 (cH6/1) virus or A/Guinea fowl/Hong Kong/WF10/99(cH9/1) viruses were generated using methods previously described (Haiet al., 2008, J Virol 82, 10580; Fodor et al., 1999, J Virol 73, 9679).Briefly, different components of the chimeric hemagglutinin (cHA) wereamplified by PCR with primers containing Sap I sites, digested with SapI, and cloned into the Sap I sites of the pDZ plasmid (Quinlivan et al.,2005, J Virol 79, 8431). For generation of the baculo-transfer plasmids,cH6/1 and cH9/1 were amplified by PCR, cut with BamHI and NotI, andcloned in frame into a modified pFastBac (Invitrogen) baculo-transfervector that harbors a C-terminal T4 phage foldon and a 6-his tag (Meieret al., 2004, J Mol Biol 344, 1051). The sequences of all plasmids wereconfirmed by Sanger sequencing.

6.4.1.2 Recombinant Baculovirus Generation, Protein Expression andPurification

In order to generate recombinant cH6/1 and cH9/1 protein,baculo-transfer vectors were transformed into E. coli strain DH10Bac(Invitrogen) according to the manufacturer's instructions. DH10Baccolonies showing the right phenotype were picked, grown up and bacmidswere prepared using a Plasmid Midi Kit (Qiagen).

Bacmids carrying the cH6/1 or cH9/1 genes were transfected into Sf9cells with Cellfectin II (Invitrogen) according to the manufacturer'sinstructions. Recombinant baculovirus was amplified in Sf9 cells grownin TNM-FH medium (Gemini Bioproducts, West Sacramento, Calif.) andtiters were determined by plaque assay (King et al., 2007, Methods MolBiol 388, 77).

High Five cells grown in HyClone SFX insect cell media (Thermo FisherScientific) were infected with recombinant baculovirus expressing cH6/1or cH9/1 at a multiplicity of infection (MOI) of 10 and a cell densityof 1×10⁶ cells/ml in 500 ml shaker flasks (Krammer et al., 2010, MolBiotechnol 45, 226). Cells were harvested 96 hours post infection andseparated from supernatant by low speed centrifugation for 10 minutes at2000 g and room temperature. For purification of cHA proteins, thesupernatant was collected and incubated with Ni-NTA resin (Qiagen) for 2hours at 4° C. The slurry was loaded onto columns and washed 3× withwashing buffer (50 mM Na2HCO3, 300 mM NaCl, 20 mM imidazole, pH 8).Protein was eluted in 0.5 ml steps with elution buffer (50 mM Na₂HCO₃,300 mM NaCl, 250 mM imidazole, pH 8), tested for protein content withBradford reagent and fractions containing protein were pooled. Pooledfractions were buffer exchanged in PBS and concentrated using an AmiconUltra centrifugal filter unit (Millipore) with a 10 kD molecular weightcut-off in a swinging bucket rotor. Protein purity and identity weretested by SDS-PAGE, Coomassie staining and Western blot. The followingantibodies were used to confirm expression of cHA: Anti-H6 goatantiserum (BEI, #NR-663), G1-26 (anti-H9, mouse; BEI# NR-9485), 3951(rabbit, anti-HA2 PR8) (Graves et al., 1983, Virology 126, 106), PY102(anti-PR8 head, mouse), and 12D1 (anti-H3 stalk, mouse) (Wang et al.,2010, PLoS Pathog 6, e1000796). Final protein concentrations weredetermined with Bradford reagent.

6.4.1.3 Rescue of Recombinant cHA Expressing Viruses

In order to rescue the recombinant virus expressing cH9/1, reversegenetics plasmids that encode vRNA and mRNA of the six wild type viralsegments from PR8, as well as plasmids encoding the N3 NA fromA/mallard/Alberta/24/01 virus and cH9/1 were used, as previouslydescribed (Hai et al., 2008, J Virol 82, 10580; Fodor et al., 1999, JVirol 73, 9679, 27). Briefly, 293T cells were transfected with 1 μg ofeach of the eight plasmids using Lipofectamine 2000 (Invitrogen)according to the manufacturer's instructions. At 12 hours posttransfection, the medium was replaced with DMEM containing 0.3% bovineserum albumin, 10 mM HEPES, and 1.5 μg of TPCK(l-1-tosylamide-2-phenylethyl chloromethyl ketone)-treated trypsin/mL(Sigma T1426). At two days post transfection, virus-containingsupernatant was inoculated into 10-day-old embryonated chicken eggs.Allantoic fluid was harvested after 2 days of incubation at 37° C. andassayed for the presence of virus by the hemagglutination of chicken redblood cells. The rescued cH9/1 virus was then propagated in 10 day oldeggs following a 48 hour incubation at 37° C. Virus stocks were titeredby plaque assay as previously described on MDCK cells in the presence ofTPCK trypsin (Steel et al., 2009, J Virol 83, 1742). The sequence of thecH9/1 RNA was confirmed by sequencing of reverse transcription-PCRproducts.

6.4.1.4 Immunofluorescence to Confirm Expression of ChimericHemagglutinin

Confluent monolayers of MDCK cells were infected with cH9/1N3 virus atan MOI of 2. At 48 hours post infection, cells were fixed and blockedwith 1% bovine serum albumin in PBS containing 0.1% Tween 20. Cells werethen incubated with mouse mAbs: anti-NP antibody HT103, PY102, anti-H9(BEI NR#9485), or a pan-H1 stalk-specific antibody (6F12). After threewashes with PBS containing 0.1% Tween 20, cells were incubated for 1hour with Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen).Infected cells were analyzed by fluorescence microscopy with an OlympusIX70 microscope.

6.4.1.5 Human Serum Samples

Human sera samples were collected and used in accordance withinstitutional protocols. Sera were collected from three patient cohorts:adults not infected with pH1N1, children not infected with pH1N1, andpH1N1 virus infected adults. Sera were collected from pH1N1 infectedpatients within the first 3 weeks of symptomatic infection. Forexperiments using pooled serum, an equal volume of each sample was mixedto generate pool. Confirmation of infection was performed by Wrammert etal. by RT-PCR and by serological assays (Wrammert et al., 2011, J ExpMed 208, 181-193).

6.4.1.6 Hemagglutinin Inhibition Assays

Collected sera were first inactivated by trypsin-heat-periodatetreatment or by RDE treatment in order to remove non-specific inhibitorsof hemagglutination (Coleman, Dowdle, 1969, Bull World Health Organ 41,415 (1969). Hemagglutination inhibition (HI) assays were performed totest reactivity with cH9/1N3 and A/duck/France/MB42/76 (H6 HA)(Desselberger et al., 1978, Proc Natl Acad Sci USA 75, 3341 (July,1978)) viruses as previously described (Lowen et al., 2009, J Virol 83,2803). A sample is considered negative if HI activity is not seen with a1:10 dilution of the serum.

6.4.1.7 ELISAs

Ninety-six well ELISA plates (Qiagen HisSorb or Nunc Immulon 2) werecoated with 50 ul of baculovirus-expressed cH6/1, cH9/1 or full lengthhemagglutinin (H5 HA, BEI NR#660; H3 HA, BEI NR#15171) proteins or H1sequence (PR8) long alpha helix (LAH) (Wang et al., 2010, Proc Natl AcadSci USA 107, 18979) diluted in PBS and incubated overnight at 4° C.Plates were blocked with 0.5% milk/2% goat sera/PBS and then washed 3×with PBS/0.1% Tween (PBST). Monoclonal antibodies, sera from humansubjects, and mouse sera used as controls were serially diluted in PBSor blocking buffer and then added to the plate, followed by a one hourincubation at room temperature. Plates were then washed 3× with PBST andincubated with 1:5000 dilution of goat anti-Human IgG [Fc] coupled toalkaline phosphatase (AP) (Meridian Life Science) or anti-mouse AP(Invitrogen) for an additional hour. Following washing 3× with PBST,p-nitrophenyl phosphate substrate (Sigma) was added. The reaction wasstopped with NaOH after 10 minutes and optical density measurements weretaken at 405 nm.

6.4.1.8 Plaque Reduction Assay

Sera from adult infected and naïve (not infected with pH1N1 virus)patients were first pooled and loaded onto a gravity flow column withprotein G sepharose (GE Healthcare) for purification of IgG proteins.The column was washed with PBS. The polyclonal antibodies were elutedwith a 0.1 M glycine buffer (pH 2.7). A 2 M Tris-HCl buffer (pH 10) wasadded immediately at a 1:10 ratio to restore the pH. The eluate wasbuffer exchanged in PBS and concentrated using an Amicon Ultra-15centrifugal filter unit (Millipore) with a 50 kD MW cut-off in aswinging bucket rotor. Protein concentrations were measured on aNanoDrop 2000 spectrophotometer using the A280 method. Removal ofnon-specific serum inhibitors was confirmed by HI tests using multiplevirus strains that demonstrated an absence of HI activity in thepurified IgG preparations (data not shown). Neutralization capability ofstalk-reactive antibodies was assessed as previously described (Wang etal., 2010, PLoS Pathog 6, e1000796). Virus was first diluted to aconcentration that would yield 100 plaque forming units per well.Different concentrations of IgG from pooled sera were then co-incubatedwith virus at room temperature for an hour. Six-well plates seeded withMDCK cells were washed with PBS and then infected with 200 ul ofvirus-IgG mixtures. Following a forty-five minute incubation at 37° C.,virus and IgG were aspirated from cells and an agar overlay containingappropriate antibody concentration and TPCK trypsin was added to eachwell. Plates were incubated for 2 days at 37° C. Plaques were visualizedby immunostaining (Bouvier et al., 2008, J Virol 82, 10052) usinganti-H9 antibody G1-26.

6.4.1.9 Pseudotype Particle Neutralization Assay

The procedure for pseudotype particle production was adapted fromprevious studies (Evans et al., 2007, Nature 446, 801). Briefly, 293-Tcells were co-transfected with four plasmids encoding (i) a pro-viruscontaining the desired reporter (V1-GLuc), (ii) HIV Gag-Pol, (iii) thechimeric cH9/1 hemagglutinin protein and (iv) influenza B/Yamagata/16/88neuraminidase (NA) (Tscherne et al., 2010, J Virol Methods 163, 336).The V1-GLuc plasmid encodes a luciferase protein that is secreted fromcells and can be detected in the cell supernatant. Supernatants werecollected 48 h post-transfection and subsequently filtered (0.45 μm poresize) in order to purify the cH9/1 particle preparations. Particles werethen incubated (at quantity determined to give luciferase activitywithin the linear range after infection) with different concentrations(50 μg/ml, 10 μg/ml and 2 μg/ml) of purified human IgGs and were addedto MDCK cells. Infections proceeded for 6 hours before cells were washedand fresh supernatant was placed over cells. All infections usingpseudotype particles were performed in the presence of 1 μg/ml polybrene(Sigma, St. Louis, Mo.) (Tscherne et al., 2010, J Virol Methods 163,336). Forty-eight hours post-infection luciferase assays were performed.

6.4.2 Results 6.4.2.1 Development of Chimeric Hemagglutinin-BasedReagents

Chimeric hemagglutinin (cHA) constructs were generated to serve asanalytical tools to assess the presence of stalk antibodies in humansera. By taking advantage of a disulfide bond that exists between C52and C277, and that delineates the boundary between the HA stalk andhead, expression plasmids were engineered that encode the globular headdomain of an H6 or H9 hemagglutinin atop the stalk domain from the HA ofPR8 virus (FIG. 14A). It was hypothesized that human serum samplescontaining stalk antibodies would likely be negative forhemagglutination inhibition (HI) activity against H6 or H9 viruses (dueto lack of prior exposure to these virus subtypes), yet would bereactive with the cH6/1 or cH9/1 constructs due to prior exposure to theH1 hemagglutinin stalk. These tools, recombinantly expressed cHAproteins and viruses expressing the cHAs, could be used to assessrelative amounts of stalk antibodies in human serum samples and tomeasure any neutralizing activity mediated by those stalk-specificantibodies.

In order to generate cH6/1 and cH9/1 protein for analytical assays,plasmids coding for the chimeric HAs were generated and expressed assoluble proteins in a baculovirus expression system. Coomassie stainingof 2 μg of total protein suggests a high degree of purity in thesepreparations (FIG. 17A). The slight delayed migration of cH9/1 isthought to be the result of an increased number of occupiedglycosylation sites on the cH9/1 head. The cHA proteins were furthercharacterized by western blot analysis using antibodies reactive withvarious parts of the HA. As shown in FIG. 17B, only cH6/1, cH9/1 andfull length HA from PR8 reacted with a rabbit polyclonal antiserumspecific for the PR8 stalk. Monoclonal antibodies against the headdomains of H6, H9, and PR8 confirmed that the chimeric constructs wereexpressing exotic heads atop a PR8 stalk. H3 protein was used as anegative control, and was only detected when using the pan-H3 antibody12D1 (Wang et al., 2010, PLoS Pathog 6, e1000796).

Recombinant viruses expressing the chimeric molecules were also rescuedfor the purpose of detecting neutralizing stalk antibodies. Because allhuman influenza viruses over the last century have encoded NA of the N1or N2 subtype, it was reasoned that the rescue of the cH9/1N3reassortant virus would allow for the assessment of the neutralizingcapability of stalk-specific antibodies, while not measuring any (N1 orN2) neuraminidase antibody activity. The cH9/1N3 virus (expressing theH1 stalk with H9 globular head HA with an N3 subtype neuraminidase) wasrescued using reverse genetics and grown to high titers in embryonatedchicken eggs. The plaque assay phenotype of this virus was similar tothat of PR8 wild type virus (FIG. 14C). To confirm the presence of theH9 head after virus passage, cells were infected with cH9/1N3 virus.Infected cells were then probed with mouse mAb G1-26, an antibodyspecific for H9 subtype hemagglutinin proteins. A pan-H1 stalk specificantibody, 6F12, was used to detect both wild type PR8 and cH9/1N3 virusinfected cells (FIG. 14B).

6.4.2.2 Stalk Specific Antibodies Bind and Neutralize cHA

In order to confirm that cH6/1 and cH9/1 proteins could be used as toolsto detect stem antibodies, the use of these cHAs was first validatedwith an antibody known to react with the HA stalk. Indeed, mouse mAbC179, an antibody reactive with the stalk of H1 HA (Okuno et al., 1993,J Virol 67, 2552), bound to baculovirus expressed cH6/1 and cH9/1protein by ELISA in a dose dependent manner (FIGS. 18A and B).

Next, it was ascertained whether replication of the cH9/1N3 virus couldbe inhibited by monoclonal antibody 6F12, which has neutralizingactivity against H1 influenza viruses (data not shown). Antibody 6F12was able to bind and neutralize cH9/1N3 virus in a plaque reductionassay (FIGS. 18 C and D), in a dose dependent manner, with one hundredpercent inhibition seen at concentrations above 4 μg/ml. These resultsvalidated the hypothesis that the chimeric proteins and the recombinantcH9/1N3 virus could be used to detect stalk antibodies with neutralizingactivity.

6.4.2.3 Patients Infected with pH1N1 have High Titers of Antibodies thatBind and Neutralize cHA

Prior to the use of cH6/1 and cH9/1 soluble proteins to quantitatestalk-reactive antibodies in patient blood samples, the sera for HIactivity was tested against viruses expressing these two HA subtypes.Using A/duck/France/MB42/76 (H6) and cH9/1N3 viruses, it was confirmedthat all adult and pediatric serum samples collected were HI negative(results not shown).

Next, the reactivity of the sera with cH6/1 and cH9/1 proteins wastested by ELISA. Sera collected from adult and pediatric subjects notinfected with pH1N1 viruses showed little reactivity with eitherprotein. However, sera collected from patients infected with pH1N1influenza virus showed enhanced binding to both cHA constructs, with agreater than 30-fold difference in IgG reactivity (comparing dilutionsthat yield equivalent optical density readings) when comparing serumpools from pH1N1 infected with those of uninfected adults and children(FIGS. 15 A and B). It was therefore reasoned, by taking the negative HIdata into account, that reactivity with cHA proteins is occurring in thestalk domain.

Using pooled samples of human sera, IgG binding to a portion of the HAstem, the long alpha helix (LAH), was also tested. These IgGs hadpreviously been shown to mediate protective immunity in mice (Wang etal., 2010, Proc Natl Acad Sci USA 107, 18979). Sera from patientsinfected with pH1N1 contained antibody reactive with the H1 LAH, whereaspatients unexposed to the pandemic virus had minimal LAH-specific serumantibody (FIG. 15C).

The H5 hemagglutinin subtype is within the same phylogenetic group asthe H1 HA, and shares a very similar stalk structure (Ekiert et al.,2009, Science 324, 246). Interestingly, patients exposed to the pH1N1had boosted serum antibody specificities reactive with the H5 protein(FIG. 15D), while not having any serum HI activity against thehomologous H5 subtype virus (data not shown). This result suggested thatexposure to the pH1N1 virus may have conferred a degree of anti-H5immunity mediated by stalk-specific antibodies.

Importantly, patients infected with pH1N1 did not have boosted serumantibody specific for an H3 hemagglutinin protein (H3 being in aseparate phylogenetic group from H1 and H5 HAs) (FIG. 15E). This resultdemonstrates that the enhanced titer of stalk-specific antibodies insera from pH1N1-infected patients is not a function of general immunestimulation; rather, the H1 stalk antibody specificities wereselectively boosted by infection with the pandemic virus strain.

Next, it was assessed whether these stalk reactive antibodies found inthese human samples had neutralizing capability. Serum samples frominfected and uninfected adults were pooled and total IgG was purified inorder to remove non-specific inhibitors (eg: sialic acid containingmolecules and lectins) that would bind to the hemagglutinin head. Usingthese pure IgG preparations, complete inhibition plaque formation atantibody concentrations above 55.5 μg/mL total serum IgG (FIGS. 16 A andB) was achieved. In accordance with the ELISA data, an approximately30-fold difference in neutralizing capability was observed whencomparing sera from pH1N1 infected with those of uninfected adults.Using mAb 6F12 as a standard, a comparison was able to be made betweenneutralizing activities mediated by 6F12 and the polyclonal human IgGpreparation. By comparing the concentrations of 6F12 and human IgGs thatyielded 100% neutralization of cHA virus, it was estimated that 7% oftotal human IgG from patients infected with pH1N1 during the last 30days comprised neutralizing stalk antibodies.

Finally, the neutralizing capability of stalk reactive antibodies wasevaluated, using a pseudotype particle infection assay that has aread-out of luciferase activity which is generated upon virus entry intohost cells. Pseudotyped particles expressing the cH9/1 protein wereincubated with purified human IgG and neutralizing activity was measuredby inhibition of particle entry resulting in absence of luciferaseenzymatic activity in cell supernatants (see methods). Consistent withthe plaque reduction assay, the pseudotyped particle assay also showed100% neutralization of particles at total IgG concentrations ofexceeding 10 μg/ml (FIG. 16C).

6.4.3 Conclusion

Novel analytical tools, in the form of chimeric hemagglutinin proteinsand viruses expressing those chimeric proteins, were developed thatallowed for the selectively detection of stalk-specific antibodies inpreparations that also include antibodies that bind the globular head ofhemagglutinin proteins. These novel hemagglutinin constructs have aconstant H1 subtype stalk, with globular head domains from distincthemagglutinin subtypes (ex: H1 stalk with H6 head). This wasaccomplished by taking advantage of a disulfide bond that exists betweencysteines 52 and 277 in the hemagglutinin protein (19) to exchange theintervening sequence with that of a different HA subtype.

Using these chimeric hemagglutinin (cHA) constructs, it was demonstratedthat a small cohort of humans with confirmed pH1N1 virus infectiongenerated a high titer of stalk specific, neutralizing antibodiescompared to uninfected adult and pediatric controls not infected withpH1N1 viruses. These findings support the hypothesis that antibodiesreactive with the hemagglutinin stalk, generated in response to pH1N1infection, likely contributed to the dying out of seasonal H1N1 virusesthat were circulating prior to the influenza pandemic of 2009.

6.5 Example 5 Influenza Viruses Expressing Chimeric Hemagglutinins:Globular Head and Stalk Domains Derived from Different Subtypes andPhylogenic Groups

This example describes several functional chimeric influenza virushemagglutinins encompassing a variety of globular head and stalkcombinations from different hemagglutinin subtypes and differentphylogenic groups as wells as recombinant influenza viruses expressingthese chimeric hemagglutinins, which had growth properties similar tothose of wild-type influenza viruses. These chimeric recombinant virusespossess growth properties similar to those of wild-type influenzaviruses and can be used as reagents to measure domain-specificantibodies in virological and immunological assays.

6.5.1 Materials and Methods 6.5.1.1 Cells and Viruses

293T and MDCK cells were obtained from the American Type CultureCollection (ATCC) and were maintained either in Dulbecco's minimalessential medium (DMEM) or in MEM (Gibco, Invitrogen) supplemented with10% fetal calf serum (HyClone; Thermo Scientific) andpenicillin-streptomycin (Gibco, Invitrogen). The A/Puerto Rico/8/1934(PR8) and A/Perth/16/2009 (Perth/09) wild type (kindly provided byAlexander Klimov, CDC) and recombinant viruses were grown in 10-day oldspecific pathogen-free embryonated hen's eggs (Charles River) at 37° C.for 2 days.

6.5.1.2 Construction of Plasmids

Plasmids encoding the different chimeric hemagglutinins were constructedusing a strategy similar to what has been previously described (see,e.g., Fodor et al., 1999, J Virol 73:9679-9682; and Hai et al., 2008, JVirol 82:10580-10590). Briefly, the different segments of chimeric HAwere amplified by PCR with primers containing SapI sites, digested withSapI, and cloned into the SapI sites of the ambisense expression vectorpDZ vector in which vRNA transcription is controlled by the human RNApolymerase I promoter and the mouse RNA polymerase I terminator, andmRNA/cRNA transcription is controlled by the chicken beta actinpolymerase II promoter (see, e.g., Quinlivan et al., 2005, J Virol79:8431-8439), through multi-segmental ligation. We kindly thank DanielPerez (University of Maryland) for the H7 HA plasmid (Genbank ID:DQ017504). The plasmids encoding A/Puerto Rico/8/1934 (PR8) genes wereused as previously described (Hai et al., 2008, J Virol 82:10580-10590).

6.5.1.3 Nucleotide Sequence Accession Number

All constructed cHA genes used in this study have been deposited in theInfluenza Research Database under the accession number IRD-RG-684014,IRD-RG-684022, IRD-RG-684030, and IRD-RG-684038. The chimeric cH1/1,cH5/1, cH7/3 and cH5/3 are listed as A/Puerto Rico/8-RGcH1-1/34,A/Puerto Rico/8-RGcH5-1/34, A/Perth/16-RGcH7-3/09, andA/Perth/16-RGcH5-3/09, respectively.

6.5.1.4 Flow Cytometric Analysis

To assess levels of hemagglutinin protein expression at the cellsurface, 293T cells were transfected with 1 μg of the appropriateplasmid using Lipofectamine 2000 (Invitrogen) according to themanufacturer's instructions or MDCK cells were infected withcHA-expressing recombinant viruses. At 48 h post-transfection, 293Tcells were trypsinized and resuspended in PBS containing 2% FBS prior tostaining with the monoclonal antibody (mAb) 6F12 (5 μg/ml), a mAbgenerated in our laboratory that is broadly reactive to the stalk domainof group 1 HAs (data not shown) or with the mAb 12D1 (5 μg/mL) againstH3 HAs (see Wang et al., 2010, PLoS Pathog 6:e1000796). At 12 hpost-infection, MDCK cells were resuspended by trypsinization andstained with the mAb 12D1. Stained cells were analyzed on a BeckmanCoulter Cytomics FC 500 flow cytometer, and the results were analyzedusing FlowJo software.

6.5.1.5 Pseudoparticle Generation and Entry Assay

The procedure for pseudoparticle production was adapted from previousstudies (see, e.g., Evans et al., 2007, Nature 446:801-805 and Tscherneet al, 2010, J Virol Methods 163:336-43). Briefly, we co-transfected293T cells with four plasmids encoding (i) a pro-virus containing thedesired reporter (V1-Gaussia luciferase) (Evans et al., 2007, Nature446:801-805), (ii) HIV Gag-Pol (Evans et al., 2007, Nature 446:801-805),(iii) chimeric hemagglutinin protein and (iv) B/Yamagata/16/88 virusneuraminidase (NA). Supernatants were collected 72 h post-transfectionand subsequently filtered (0.45 μm pore size). The presence ofpseudotype virus like particles (VLPs) was evaluated throughhemagglutination assay. Different VLP preparations were adjusted to thesame 4 hemagglutination units prior to inoculation of MDCK cells. All ofthe following assays using pseudoparticles were performed in thepresence of 1 μg/mL polybrene (Sigma) to increase the efficiency oftransduction (see, e.g., Evans et al., 2007, Nature 446:801-805 andTscherne et al, 2010, J Virol Methods 163:336-43).

The entry assay was performed by transducing MDCK cells withpseudoparticles that expressed different chimeric hemagglutinins andcontained the Gaussia luciferase reporter. Twenty-four hourspost-transduction, cells were washed three times with fresh medium toremove any residual Gaussia luciferase protein present in the inoculum.Forty-eight hours post-transduction, luciferase assays were performed(Evans et al., 2007, Nature 446:801-805).

6.5.1.6 Rescue of Recombinant Chimeric Influenza A Viruses

Rescue of influenza A viruses from plasmid DNA was performed aspreviously described (see, e.g., Fodor et al., 1999, J Virol73:9679-9682; and Hai et al., 2008, J Virol 82:10580-10590). To generatethe recombinant wild-type (rWT) PR8 virus, 293T cells wereco-transfected with 1 μg of 8 pDZ PR8 rescue plasmids usingLipofectamine 2000 (Invitrogen). The wild type HA plasmid wassubstituted with a plasmid encoding the desired chimeric HA in order togenerate cHA-expressing recombinant viruses. At 6 h post-transfection,the medium was replaced with DMEM containing 0.3% bovine serum albumin(BSA), 10 mM HEPES, and 1.5 μg/ml TPCK (L-1-tosylamide-2-phenylethylchloromethyl ketone)-treated trypsin (Sigma). After 24 hourspost-transfection, 8-day old embryonated chicken eggs were inoculatedwith virus-containing supernatant. Allantoic fluid was harvested after 2days of incubation at 37° C. and assayed for the presence of virus byhemagglutination of chicken red blood cells. Virus stocks were titeredby plaque assay on MDCK cells as previously described (Fodor et al.,1999, J Virol 73:9679-9682, Hai et al., 2008, J Virol 82:10580-10590).

6.5.1.7 Virus Growth Kinetics Assay

To analyze the replication characteristics of recombinant viruses,10-day old embryonated chicken eggs were inoculated with 100 plaqueforming units (pfu) of wild-type or cHA-expressing recombinant viruses.Allantoic fluid was harvested and subsequently assayed for viral growthat 0, 9, 24, 48, and 72 h post-infection (hpi). The titers of viruspresent in allantoic fluid were determined by plaque assay on MDCK cellsas referenced above.

6.5.1.8 Immunostaining of Plaques

Plaques were visualized by immunostaining with mAb HT103 against theinfluenza A nucleoprotein (NP), using a protocol that has beenpreviously described (Bouvier et al., 2008, Journal of Virology82:10052-8; and Steel et al., 2009, J Virol 83:1742-53).

6.5.1.9 Western Blot and Indirect Immunofluorescence Analysis

Confluent MDCK cells were infected (multiplicity of infection [MOI] of2) with indicated recombinant influenza viruses or mock infected withphosphate-buffered saline (PBS) for 1 h at 37° C. At 12 hpi, cells werelysed in 1×SDS loading buffer as described previously (see, e.g., Hai etal., 2008, J Virol 82:10580-10590). The reduced cell lysates wereanalyzed by Western blot analysis using monoclonal antibodies (mAbs)against influenza A virus nucleoprotein (NP) (HT103), PR8 HA head domain(PY102), Cal/09 HA head domain (29E3), VN/04 HA head domain (mAb #8) and12D1, a pan-H3 antibody reactive against the HA stalk. In order todetect H7 head domains, polyclonal goat sera NR-3152 was used (raisedagainst A/FPV/Dutch/27 (H7) virus, BEI Resources). Ananti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Abcam)was used for the loading control. Proteins were visualized using anenhanced chemiluminescence protein detection system (PerkinElmer LifeSciences).

For immunofluorescence analysis, confluent monolayers of MDCK cells on15-mm coverslips were infected with recombinant viruses at an MOI of 2.At 15 hpi, cells were fixed and permeabilized with methanol-acetone(ratio, 1:1) at −20° C. for 20 minutes. After being blocked with 1%bovine serum albumin in PBS containing 0.1% Tween 20, cells wereincubated for 1 h with the antibodies described above, as well as mAb6F12. After three washes with PBS containing 0.1% Tween 20, cells wereincubated for 1 h with Alexa Fluor 594-conjugated anti-mouse IgG(Invitrogen) or Alexa Fluor 594-conjugated anti-goat IgG (Invitrogen).Following the final three washes, infected cells were analyzed byfluorescence microscopy with an Olympus IX70 microscope.

6.5.1.10 Plaque Reduction Assay

Plaque reduction assay was performed as previously described (see Wanget al., 2010, PLoS Pathog 6:e1000796). Approximately 60 to 80 pfu ofrecombinant viruses expressing cHA made up of Cal/09 or VN/04 globularhead domains atop a PR8 stalk were incubated with or without differentconcentrations (100, 20, 4, 0.8, 0.16 and 0.032 ug/ml) of mAb KB2, abroadly neutralizing anti-HA stalk antibody generated in our laboratory(data not shown) for 60 minutes in a total volume of 240 uL at roomtemperature. A confluent layer of MDCK cells in E-well plates was washedtwice with PBS and then incubated with the antibody-virus mixture for 40minutes at 37° C. A TPCK-trypsin agar overlay supplemented with antibodyat the above-described concentrations or no antibody was then added toeach well after the inoculum had been aspirated off. Plates wereincubated for 2 days at 37° C. Plaques were then visualized byimmunostaining (Bouvier et al., 2008, Journal of Virology 82:10052-8;and Steel et al., 2009, J Virol 83:1742-53) with anti-influenza ANPantibody HT103.

6.5.1.11 Pseudotype Particle Neutralization Assay

The procedure for pseudotype particle production was the same asdescribed above, using the cHA construct that is comprised of either aVN/04 (H5) or a Cal/09 (H1) head and a PR8 (H1) stalk with the influenzaB/Yamagata/16/88 virus NA. Particles were then incubated with differentconcentrations of mAb KB2 at 5 fold dilutions from 100 to 0.032 μg/mL.Then, these mixtures were added to MDCK cells. Transductions proceededfor 6 hours before cells were washed and fresh medium was placed overcells. All transductions using pseudotype particles were performed inthe presence of 1 μg/mL polybrene (Sigma, St. Louis, Mo.) (Tscherne etal, 2010, J Virol Methods 163:336-43). Forty-eight hourspost-transduction, luciferase assays were performed in order to assaythe degree in which entry was blocked by mAb KB2.

6.5.2 Results 6.5.2.1 Generation of Chimeric Hemagglutinins

In order to see if the cysteine residues forming the Cys52-Cys277disulfide bond were conserved, an alignment of influenza A virus HAsequences of the H1, H3, H5 and H7 subtypes were used in this study.Because these cysteine residues are highly conserved across HA subtypes,for both group 1 and group 2 HAs, the Cys52-Cys277 disulfide bond wasused as a delineating point between the head and stalk domains. Bydefining the sequence between Cys52 and Cys277 as the head region, andthe remainder of the molecule as the stalk, it was rationalized thatconstructs could be made that encode novel head and stalk combinationsfrom a variety of HA subtypes (FIGS. 19 A and B).

The degree of amino acid identity that exists between the stalk regionsof hemagglutinin subtypes further encouraged us that the swapping ofhead domains might be possible. Higher percentages of amino acididentity were seen in the stalk domains across all subtypes, compared tothe head domains (FIG. 20).

All 16 subtypes of influenza HA are classified into two phylogeneticgroups (Palese and Shaw, 2006, Orthomyxoviridae: the viruses and theirreplication, Fields virology, 5th ed., 1647-1690). Because higherpercentages of amino acid identity was observed within stalk regions ofa particular group (FIG. 20), and because one cHA virus that containedhead and stalk domains from group 1 viruses had been successfullygenerated (Pica et al., 2012, PNAS 109:2573-8), an attempt was made togenerate intra-group cHAs. For group 1, two chimeric hemagglutininconstructs that encode either the pandemic H1 Cal/09 HA or VN/04globular head domain with the stalk region from PR8 (H1) HA (cH1/1 andcH5/1, respectively) were generated (FIG. 19B). A similar strategy wasapplied to generate a chimeric HA that expressed head and stalk domainsfrom different group 2 influenza strains: the head from Alb/01 (H7,group 2) and the stalk region from Perth/09 (H3, group 2) HA (cH7/3)(FIG. 19B). Finally, it was evaluated whether the head and stalk domainscould be swapped to make an inter-group chimeric HA containing the headdomain of VN/05 HA (H5, group 1) atop a Perth/09 HA (H3, group 2) stalk(cH5/3) (FIG. 19B).

Following the construction of these plasmids, experiments were performedto determine whether the different chimeric HA constructs could beexpressed and transported to the cell surface like wild-type HAs.Fluorescence-activated cell sorter (FACS) analysis of transientlytransfected 293T cells was performed following surface staining with H1and H3 stalk domain specific antibodies, respectively. Using thismethod, cell surface expression of all four chimeric constructs weredetected (FIG. 21). However, compared to the wild type PR8 HA lesssurface protein expression was detected for the cH1/1 construct, whichcould be attributed to the inherent character associated with the headdomain of the Cal/09 HA or a lower transfection efficiency for thischimeric DNA construct. In addition, it is of note that there weredifferences in the cell surface expression pattern for the cH7/3 andcH5/3 constructs. This “double peak” expression pattern was observedonly in transfection conditions, and was reproducible. It was notdetected upon infection with either cH7/3 or cH5/3-expressingrecombinant viruses (FIG. 21). Therefore, these data indicate that thecHAs can be transported through the Golgi complex to the cell surface.

Next, the entry characteristics of the different cHAs throughtransduction of MDCK cells were examined using retroviral pseudotypeparticles that contained a luciferase reporter construct and expressedthe cHA and wild-type B/Yamagata/16/88 virus NA on the particle surface.The entry efficiency mediated by the cHA proteins was detected by theluciferase read-out. Comparable levels of pseudotype particle-mediatedluciferase expression were observed for cH5/1, cH7/3 and cH5/3 chimericHAs and the corresponding wild type proteins (FIG. 22). Particlesencoding the cH1/1 HA expressed lower luciferase levels compared to theother HA constructs, which could be due to either the lower expressionof the cH1/1 in the producer cell line and hence fewer HA trimers perparticle or the less efficient entry properties of the cH1/1 HA. It isalso possible that when normalizing the pseudotype particles to 4hemagglutinin units, the actual amount of pseudotype particles may varydue to differences in binding to red blood cells.

6.5.2.2 Generation of Recombinant Influenza Viruses Bearing ChimericHemagglutinins

Because it had determined that our cHA constructs were efficientlyexpressed and transported to the cell surface, a study was performed toassess whether a recombinant influenza virus that encodes a cHA could berescued. Viruses containing the different cHAs were successfullygenerated using previously published protocols (see, e.g., Fodor et al.,1999, J Virol 73:9679-9682; and Hai et al., 2008, J Virol82:10580-10590). The resulting viruses were plaque purified, amplifiedin 10 day old embryonated eggs and the chimeric segments were analyzedby RT-PCR and sequenced. In all cases, the virus was found to have theexpected chimeric HA segment and no other HA segment (data not shown).

The presence of the cHAs in rescued viruses was further confirmed byWestern blot (FIG. 23) and indirect immunofluorescence of infected cells(FIG. 24). MDCK cells were infected with rWT PR8, wild-type Perth/09,cH1/1, cH5/1, cH7/3 and cH5/3 viruses. cH1/1 and cH5/1 chimeric HAproteins were detected in the corresponding samples using antibodiesreactive against the head domains of Cal/09 (H1) HA (29E3) (Medina etal., 2010, Nature Communications 1:28) or VN/04 (H5) HA (mAb #8) (Steelet al., 2009, J Virol 83:1742-53) respectively (FIG. 23). Comparableexpression levels among the cH7/3, cH5/3 and wild type Perth HA wereobserved using 12D1, a pan-H3 anti-stalk mAb (see Wang et al., 2010,PLoS Pathog 6:e1000796). The wild type Perth HA showed a slowermigration on the gel that is likely due to a higher number ofglycosylation sites in the globular head domain. It was confirmed thatthe correct HA head domain was expressed atop an H3 stalk by usinganti-H7 polyclonal (NR-3152) or anti-H5 monoclonal antibodies (mAb #8)on cH7/3 or cH5/3 infection samples, respectively. Positive bands weredetected in both cases.

For the immunofluorescence study, the infection conditions were similarto those used for Western blot analysis. Infected cells were stainedwith corresponding antibodies as used in FIG. 23. All infected cellsshowed the expected expression of the chimeric and wild type HAs, aswell as of the influenza A virus NP (FIG. 24).

6.5.2.3 Replication Characteristics of Recombinant Viruses

The growth properties of wild type and recombinant viruses were assessedin 10-day-old embryonated chicken eggs at 37° C. (FIG. 25A). The rWT PR8virus was included for comparison of the growth kinetics of therecombinant viruses expressing chimeric HAs. cH5/1 and cH5/3 virusesdisplayed comparable replication kinetics to that of rWT PR8 virus.cH7/3 virus grew to similar peak titers as rWT PR8 at 48 hpi (1×10⁹PFU/mL), though there was a 2 log reduction in viral titer compared tothe rWT PR8 virus at 9 hpi. The cH1/1 virus was attenuated as comparedto the rWT PR8 virus, as shown by reduced viral titers at all timepoints. Nonetheless, cH1/1 virus reached a respectable peak titer ofapproximately 10⁸ PFU/mL. The Perth/09 Wild type virus grows tocomparable peak titers in embryonated eggs (data not shown).

The plaque phenotype of each of the chimeric viruses was also evaluatedin MDCK cells. All viruses formed comparable sized plaques as shown inFIG. 25B. These data together confirm that the chimeric HA constructsfold correctly and are biologically functional.

6.5.2.4 Stalk Specific Antibodies can Neutralize cHA-Expressing Virusesand Pseudoparticles

Finally, stalk-specific antibodies were tested for the ability toneutralize our newly generated recombinant viruses expressing cHAs.Plaque reduction assays were performed in the presence of mAb KB2, anHA-stalk specific antibody with broad group 1 reactivity or withoutantibody. It was shown that mAb KB2 neutralizes all cHA-expressingviruses with similar efficiency and in a dose dependent manner. At 100ug/mL, mAb KB2 was able to completely neutralize cH1/1 and cH5/1 viruseswith 100% efficiency, with some neutralizing activity at concentrationsas low as 4 ug/mL (FIG. 26A).

To confirm these results, a pseudotype particle inhibition assay wasperformed with mAb KB2. Pseudotype particles expressing cH1/1 or cH5/1and influenza B virus NA were added to MDCK cells in the presence of mAbKB2, or without antibody. Forty-eight hours post-transduction,supernatant was collected and luciferase activity was analyzed. Asexpected, mAb KB2 blocked the entry of cH1/1 and cH5/1 pseudotypeparticles in a dose dependent manner at concentrations above 4 ug/mL.While a lower concentration of mAb KB2 was sufficient to inhibit entryof pseudotype particles compared to concentrations used in the plaquereduction assay, this was an expected result due to the assumed lowerincorporation of HA trimers on the surface of pseudotype particles(Corti et al., 2010, The Journal of Clinical Investigation 120:1663-73).This phenomenon of different neutralizing potencies of mAbs in assaysthat involve whole virus versus pseudotype particles has beenappreciated in other studies (Corti et al., 2010, The Journal ofClinical Investigation 120:1663-7321; Sui et al., 2009, NatureStructural & Molecular Biology 16:265-73).

6.5.3 Conclusion

A novel strategy was developed to generate influenza viruses withchimeric HA proteins bearing different HA globular head domains bytaking advantage of the conserved disulfide bond Cys52-Cys277 whichdemarcates the border between the head and stalk domains. Thus, throughsubstituting the parental head domain with the head domain of anotherHA, a panel of chimeric HAs with the same stalk but different globularheads was generated. The design was tested across multiple subtypes,including the PR8 stalk domain with Cal/09 and VN H5 globular heads. Inaddition, an H7 globular head was placed on an H3 stalk domain. Theseconstructs cover both phylogenetic groups of the influenza HA protein.Each construct was expressed on the cell surface and retained fusionactivity. The generation of recombinant viruses bearing the chimeric HAsfurther validated that the HAs fold correctly and retain biologicalfunctions.

6.6 Example 6 Chimeric Hemagglutinin Constructs as a Universal InfluenzaVaccine

This example demonstrates the protective efficacy of a stalk-specificimmune response that can be elicited through vaccination with chimerichemagglutinin (cHA) constructs, proteins that contain uniquehemagglutinin head and stalk combinations.

6.6.1 Materials and Methods 6.6.1.1 Cells and Viruses

293T and MDCK cells were obtained from ATCC and were maintained inDulbeccos's Modified Eagle's medium (DMEM) and Minimal Essential Medium(both from Gibco). Each were supplemented with 10% fetal calf serum(HyClone), and 100 units/ml of penicillin-100 μg/ml of streptomycin(Pen/Strep, Gibco).

Influenza virus A/Fort Monmouth/1/1947 (FM1) and A/Netherlands/602/2009were passaged in mouse lungs and then grown in 10 day old embryonatedchicken eggs for 48 hours. Low pathogenicity A/Vietnam/1203/04(VN04):PR82:6 reassortant virus with the polybasic cleavage site removed(see, e.g., Steel et al., 2009, J Virol 83:1742-1753) andB/Yamagata/16/1988 virus were grown in 10-day old embryonated eggs for48 hours at 37° C. or 72 hours at 33° C., respectively.

Recombinant influenza viruses were produced by reverse genetics systemas described above and as previously described (see, e.g., Quinlivan etal., 2005, J Virol 79:8431-8439). cH9/1 N1 virus, a virus expressing theHA globular head domain of an H9 virus atop an H1 stalk (from PR8virus), and cH5/1 (H5 head (VN04), H1 stalk) N1 viruses were rescued ina similar manner as previously described (see, e.g., Pica et al., 2012,PNAS USA 109:2573-2578). In order to generate the YAM-HA virus, theextracellular domain of the B/Yamagata/16/1988 (WT YAM) HA wassubstituted with the corresponding domain of A/Puerto Rico/8/1934 virusHA (see, e.g., Hai et al., 2011, Journal of virology 85:6832-6843). Thereverse genetic plasmids encoding the other 7 WT YAM viral segments wereconstructed in a previous study (see, e.g., Hai et al., 2008, J Virol82:10580-10590). Following rescue, cHA-expressing recombinant viruseswere propagated in 10 day old embryonated chicken eggs for 48 hours at37° C. YAM-HA virus was grown in 8-day old embryonated chicken eggs for72 hours at 33° C.

Recombinant and wild-type viruses were titered on MDCK cells (ATCC) inthe presence of TPCK trypsin as described above. cH5/1 N1 virus waspartially purified over a 30% sucrose cushion for use in ELISA assays.cH9/1 N1, cH5/1 N1 and FM1 viruses were purified via gradientcentrifugation and inactivated with formaldehyde diluted (1:4000) in PBSto be used as positive control vaccines.

6.6.1.2 Generation of cH6/1 and cH9/1 Protein Constructs

Soluble cH6/1 and cH9/1 proteins were generated using a baculovirusexpression system as described above and as previously described (see,e.g., Pica et al., 2012, PNAS USA 109:2573-2578). Briefly,baculotransfer vectors were first generated followed by transfection ofbacmids into Sf9 cells. Recombinant baculovirus were then used to infectHigh Five cells at an MOI of 10. Supernatants were harvested 96 hpostinfection and then incubated with Ni-NTA resin (Qiagen) for 2 h at4° C. to purify His-tagged recombinant cHA proteins. The slurry wasloaded onto columns, and following washes, was eluted in pH 8 elutionbuffer (50 mM Na2HCO3, 300 mM NaCl, 250 mM imidazole). Pooled fractionsthat contained protein were buffer-exchanged in PBS and concentratedusing an Amicon Ultra centrifugal filter unit (Millipore) with a 10-kDamolecular mass cutoff in a swinging bucket rotor. Protein purity andidentity were tested by SDS/PAGE, Coomassie staining, and Western blot.Final protein concentrations were determined with Bradford reagent.

6.6.1.3 Animals

Animals were allowed access to food and water ad libitum and kept on a12 hour light/dark cycle. Female 6-8 week old BALB/c mice (JacksonLaboratories) were anesthetized for all intranasal procedures withintraperitoneal (IP) injection of 0.1 ml of ketamine/xylazine (0.15 mgketamine and 0.03 mg xylazine).

6.6.1.4 Vaccination and Challenge Experiments

Naïve 6-8 week old female BALB/c mice were vaccinated with cH9/1protein, intranasally (10 ug) in the presence of adjuvant R848(Invitrogen) and intraperitoneally (10 ug) with Addavax, an MF59-likeadjuvant (Invitrogen). Animals were boosted with cH6/1 protein, or BSA(BioRad) three weeks post prime. Booster vaccinations were alsoadministered intranasally (10 ug) and intraperitoneally (10 ug), thoughwith poly I:C as an adjuvant (Invitrogen). Inactivated FM1 virus (1 ug)was administered intramuscularly in a volume of 50 ul as a positivecontrol. Three weeks post boost, animals were bled and sera washarvested, and animals were challenged with 5 LD50 of FM1 virus. Weightswere monitored for 14 days post challenge.

In other experiments, animals were primed with cH9/1 encoding plasmidDNA (80 ug, TriGrid delivery system; Ichor Medical Systems) and thenboosted three weeks later with cH6/1 or cH9/1 (control) proteinadministered with polyI:C intranasally (10 ug) and intramuscularly (10ug). The boost was repeated three weeks later with cH5/1 or cH9/1(control) protein. Control animals were DNA electroporated with cH9/1coding DNA as well but were boosted twice with BSA (in a similar way asthe treatment group). Positive control animals received eitherinactivated FM1 or PR8 virus (1 μg) or 1 μg of pH1N1 monovalent splitvaccine intramuscularly (BEI). Animals were then challenged 3-5 weekspost boost with 5 LD50 of PR8 or FM1 or 10 LD50 of pH1N1 virus. Animalsused for CD8+ T-cell depletion were treated 48 and 24 hours prior tochallenge with 300 μg of a anti-CD8+ T-cell antibody (see, e.g., M. L.Salem, 2000, Int. J. Immunopharmacol. 22:707) (from hybridoma line 2.43,purchased from ATCC) and challenged with 5 LD50 of PR8 virus. Weightswere monitored for 14 days post challenge. A 20% cutoff was used for allviruses.

For other experiments, mice were inoculated with YAM-HA or WT YAM virus,and then vaccinated with BSA or cH6/1 protein in the presence of polyI:C intramuscularly and intraperitoneally three weeks later. Naïveanimals served as an additional control. All animals were bled andchallenged 3-5 weeks following vaccination with 250 LD50 cH9/1 N1 virus,or 10LD50 of 2:6 reassortant H5 virus with the polybasic cleavage siteremoved in the PR8 background (see, e.g., Steel et al., 2009, J Virol83:1742-1753). Formaldehyde-inactivated cH9/1 N1 virus (1 ug) and cH5/1N1 virus were administered intramuscularly in a volume of 50 ul as apositive control for the appropriate viral challenge. Animals wereeuthanized if they lost more than 30% of their initial body weightfollowing challenge, according to institutional guidelines. A 20% cutoffwas used for infection with the less pathogenic cH9/1 N1 andA/Netherlands/602/2009 viruses. For challenges using these two viruses,more stringent doses were used in order to appreciate death orsubstantial weight losses of all controls (250 or 10 LD50).

6.6.1.5 Enzyme Linked Immunosorbant Assay

Immulon 4HBX (Thermo Scientific) plates were coated overnight withpartially purified cH5/1 N1 virus diluted to 5 ug/mL in PBS. Plates wereblocked for 1 hour with 0.1% Tween 20-PBS (TPBS) containing 3% non-fatmilk powder, and then incubated with mouse sera serially diluted in TPBScontaining 1% milk powder for 1 hour at room temperature. After threewashes, plates were incubated for 1 hour at room temperature withalkaline phosphate (AP) linked anti-mouse IgG (γ-chain specific,Invitrogen). Plates were then washed three times with TPBS, developedwith p-nitrophenylphosphate (PNPP) substrate (Zymed), stopped with 0.5MNaOH, and read at the optical density of 405 nm. For all experiments, aSynergy 4 (BioTek) plate reader was used.

Stalk-specific antibody titers were detected by ELISA as describedabove. PR8 antigens were used to coat ELISA plates in order to quantifystalk-specific reactivity. To detect the neutralization capability ofstalk-specific antibodies in vaccinated mice, sera were pooled and totalserum IgG was purified.

Pseudoparticles expressing H5 HA were used in a pseudoparticle entryassay as previously described (see, e.g., Hai et al., 2012, J. Virol.86:5774-5781 and N. Pica et al., 2012, PNAS 109:2573-2578). Upon entry,pseudoparticles express a luciferase reporter Inhibition of this entryby IgG was quantified as the percentage of expression compared tonon-IgG treated controls. Because animals had not been exposed to H5globular head domains, these assays determined the degree to whichstalk-specific antibodies produced by vaccination neutralize virus.Monoclonal antibody CR6261 and purified IgG from influenza B wild typeinfected mice were used as controls.

6.6.1.6 Plaque Reduction Neutralization Assay (PRNA)

Dilutions of mAbs were first pre-incubated with 60 to 80 plaque-formingunits (pfu) of virus (cH9N1 influenza A virus) for 1 hour at RT on ashaker. The virus and purified IgG mixture was then used to infect amonolayer of MDCK cells in duplicate in a 12-well format and incubatedat 37° C. for 1 hour with intermittent rocking every 10 minutes. Theagar overlay was supplemented with corresponding IgG dilutions. At twodays post infection (dpi), the monolayer was fixed with 4% PFA/1×PBS for30 minutes. Cells were blocked with 5% NF-milk/1×PBS for 30 minutes atRT and were incubated accordingly with either with an H9 specificmonoclonal antibody (5 μg/mL) for 1 hour at RT. An anti-mouse secondaryconjugated to HRP was used as a secondary at a 1:1000 dilution. Plaqueswere visualized using TrueBlue peroxidase substrate (KPL Inc.) and thereaction was stopped with tap water. Plaques were counted for eachantibody and percent inhibition calculated over the no mAb group.

6.6.1.7 Statistical Tests

Statistical analyses were performed using a one tailed student's T test(Prism4, GraphPad). For FIG. 29C, all values are plotted as averageswith standard error of the mean. Differences in survival were calculatedwith Kaplan Meier survival analysis with log rank significance test.

For analyses with P-values, P-values at or below 0.05 are consideredstatistically significant. Welch's correction was used if variances weredetermined to be statistically different. P-values at or below 0.05 areconsidered statistically significant. When comparing stalk serumreactivity to maximum weight loss in FIG. 29C, one value was detected asan outlier (modified Z-score >3.5 standard deviations above the mean)according to the methods of Iglewicz and Hoaglin (see Iglewicz, B. a.H., D. 1993. Volume 16: How to detect and handle outliers. In E. Mykytka(ed.), The ASQC Basic References in Quality Control: StatisticalTechniques. American Society of Quality Control), and was omitted fromanalyses.

6.6.2 Results 6.6.2.1 Sequential Vaccination with cHA Constructs ElicitsHA Stalk-Specific Antibodies and Provides Protection from LethalInfluenza Challenge

It was hypothesized that constructs that express globular head domainsfrom viruses with different antigenicities could stimulate polyclonalresponses towards the stalk domain of the HA. To test this, mice werefirst vaccinated with cH9/1 soluble protein with adjuvant, whereby thestalk of the HA is from A/Puerto Rico/8/1934 (PR8) virus and the headfrom an H9 isolate. Three weeks post prime, mice were boosted with asecond soluble cHA, cH6/1 (head from H6 virus, stalk from PR8 virus),with the intent of stimulating humoral responses towards the stalkdomain of the molecule. (Mice vaccinated with inactivated FM1 virusserved as a positive control). Three weeks post boost, mice were bled toassess serum reactivity to the H1 stalk domain, and then challenged withmouse-adapted A/Fort Mounmoth/1/1947 (FM1) virus. As shown in FIG. 27A,vaccinated mice produced serum antibody responses towards the HA stalkdomain. Following challenge with FM1, animals lost a considerable amountof weight (FIG. 27B), though recovered after day 7 for an overallsurvival rate of 90% (FIG. 27C). Even though mice had only been exposedto the globular head domains of H9 and H6 viruses, it was verified thatall mice were HI negative to FM1 virus, and thereby confirmed that theprotection elicited from vaccination was the result of an immuneresponse specific to the stalk domain. Therefore, vaccination withPR8-based cHAs provides stalk-specific immunity that is protective inthe face of an FM1 virus challenge.

6.6.2.2 Vaccination with cH6/1 Protein Elicits Stalk-Specific Immunitythat Mediates Protection from cH9/1 N1 Virus Challenge

Although antibody responses were generated towards the stalk byadministering two different soluble cHA constructs, a substantial degreeof morbidity was seen following FM1 challenge. Because mice areimmunologically naïve to influenza virus, it was possible that multipleexposures to influenza virus followed by the introduction of anantigenically distinct head was required in order to induce high serumantibody titers against the HA stalk. Enhanced stimulation of serumantibody titers with specificity to the hemagglutinin stalk may alsorequire infection, and may explain why a robust protection followingprime and boost with cHA proteins alone was not observed.

In order to stimulate immune responses towards the viral hemagglutinin,but not generate protective immunity to other viral proteins, arecombinant B/Yamagata/16/1988 virus was constructed that expresses theectodomain of the HA from PR8 virus (YAM-HA) (see, e.g., Hai et al.,2011, Journal of virology 85:6832-6843). Mice were inoculated withYAM-HA in order to mimic prior exposures to influenza virus, and thenvaccinated 3 weeks later with BSA or cH6/1 protein. As an additionalcontrol, mice were infected with wild-type B/Yamagata/16/1988 (WT YAM)virus and vaccinated with BSA. An influenza A virus that expressed thecH9/1 (H9 head, H1 stalk) was then used as the challenge virus, in orderto definitively demonstrate the protective nature of an immune responsedirected only towards the HA stalk. Again, because animals were exposedto the globular head domains from H1 and H6 viruses, the protection seenfollowing challenge with a virus that expressed the cH9/1 HA was mostlikely a result of immunity towards the H1 stalk domain.

As shown in FIG. 28A, animals that received cH6/1 protein vaccinefollowing YAM-HA exposure were completely protected from 250 LD50challenge with a cH9/1 expressing virus in the PR8 background. Animalsvaccinated with cH6/1 soluble protein lost statistically less weight ondays 3, 4, and 5 compared to animals that were vaccinated with BSA. Thisprotection from weight loss resulted in increased survival in the groupvaccinated with cH6/1, compared to the cohort vaccinated with BSA(p=0.038; FIG. 28B). Naïve animals and those inoculated with WT YAM werenot protected from infection, demonstrating that any protection that wasseen in the other vaccination groups was not a result of viralreplication, but was instead a specific response to the H1 stalk domain.Because animals were exposed to the globular head domains from H1 and H6viruses, and were HI negative to the cH9/1 challenge virus, it isbelieved that the protection seen here is a result of immunity towardsthe H1 stalk domain.

It is of note that monoclonal antibodies with specificities to the HAstalk have been isolated from individuals infected with or vaccinatedagainst seasonal H1N1 viruses (see, e.g., Corti et al., 2010, TheJournal of clinical investigation 120:1663-1673; Corti et al., 2011,Science 333:850-856; Ekiert et al., 2011, Science 333:843-850; Sui etal., 2009, Nat Struct Mol Biol 16:265-273; Throsby et al., 2008, PLoSOne 3:e3942), and stalk titers have been appreciated in individuals notinfected with the pH1N1 virus, although at lower levels (see, e.g., Picaet al., 2012, PNAS USA 109:2573-2578). As such, it is not surprisingthat YAM-HA inoculated animals were able to generate some degree ofstalk titer. Vaccination with the cH6/1 construct, however, increasedserum stalk titers by 4 fold (reciprocal dilutions that yieldedequivalent OD values) (FIG. 28C, and protected animals from substantialweight loss and death (FIGS. 28A and 28B). Vaccination with the cH6/1construct elicited the production of stalk-specific IgG that neutralizedvirus with 100% efficiency (YAM-HA+BSA) (FIG. 28D) whereas serum fromprime only animals exhibited neutralizing levels barely above background(YAM-HA+BSA). Indeed, animals inoculated with YAM-HA and then vaccinatedwith BSA had statistically similar survival rates to those that wereinoculated with WT YAM virus and vaccinated with BSA (p=0.058). Incontrast, the use of cH6/1 protein as a vaccine yielded 100% survivalfrom challenge, a rate that was highly significant when compared to thatof animals inoculated with WT YAM (p<0.0001). Survival was also enhancedwhen compared to that of mice inoculated with YAM-HA and vaccinated withBSA (p=0.038). These differences were not reflected in thepseudoparticle entry assay, as IgG from YAM-HA+BSA mice and YAM-HA+cH6/1mice inhibited the entry of pseudoparticles encoding an H5 HA withsimilar efficiency (FIG. 28E). It is important to note that the latterassay only detects the ability of antibodies to block entry ofpseudoparticles. Therefore, the effects of stalk-antibodies downstreamof entry and/or their interaction with infected (immune) cells would notbe detected in this assay. This might explain why differences inneutralization were not observed between the two groups and did notreflect the in vivo findings. Nonetheless, the antibodies elicited bythese infection protocols were stalk specific and were broadlyneutralizing. Because the challenge virus only encodes the stalk domainfrom an H1 virus, it can be concluded that the protection seen was theresult of the host immune response to the HA stalk domain that wasstimulated through cH6/1 vaccination.

6.6.2.3 Vaccination with cH6/1 Protein Protects Mice from Lethal H5Influenza Virus Challenge

Whether vaccination with cH6/1 protein could protect mice from challengewith an H5 virus was next ascertained. Mice were inoculated andvaccinated as described above, and challenged with 10 LD50 of a 2:6reassortant virus that expresses the HA and NA from A/Vietnam/1203/2004virus in the PR8 background (see, e.g., Steel et al., 2009, J Virol83:1742-1753). As expected, naïve animals and those inoculated with WTYAM virus were not protected from challenge and succumbed to infectionby day 8. Animals inoculated with YAM-HA virus and vaccinated with BSAwere marginally protected from challenge, with a survival rate of 40%.Increased protection was seen when animals were vaccinated with cH6/1protein, with 90% survival. The difference in survival rates between thetwo vaccine groups approached statistical significance (p=0.06),although mice vaccinated with cH6/1 protein survived for a staticallylonger time (p=0.037) (FIGS. 29A and 29B). When comparing reactivity tothe HA stalk to the % maximal weight loss over the monitoring periodfollowing H5 challenge, an inverse correlation was detected, wherebyanimals with higher serum stalk titers tended to lose less weightfollowing challenge (FIG. 429C), supporting the notion that cH6/1 canboost HA-stalk based immunity.

Using challenge viruses with HA globular head domains to whichvaccinated mice were immunologically naïve and HI negative, the resultsindicate that protection from challenge following vaccination was solelybased on an immune response towards the HA stalk. To exclude thepossibility that cross reactive antibodies towards the receptor bindingsite could be playing a role in the protection seen here, mice were alltested for HI and found to be HI negative to their respective challengeviruses.

6.6.2.4 Vaccination with cHA Elicits Stalk-Specific Immunity thatMediates Protection from H1N1 Virus Challenges

Stalk-specific antibodies have been detected in human sera (see, e.g.,FIGS. 15A and 15B; M. Thorsby et al., 2008, PLoS One 3:e3942, D. C.Ekiert et al., 2009, Science 324:246-251, D. C. Ekiert et al., 2011,Science 333:843-850, J. Wrammert et al., 2011, J. Exp. Med. 208:181-193and N. Pica et al., 2012, PNAS 109:2573-2578). Because it is possiblethat previous exposure to influenza virus HA is critical to the robustproduction of a stalk specific immune response, it was ascertainedwhether preexisting immunity to the influenza virus in mice could berecapitulated. It was hypothesized that this would more effectivelyprotect against morbidity following virus challenge. To achieve this,mice were primed with a DNA expression vector (see, e.g., J. Steel etal, 2010, M Bio 1(1), pii:e00018-10) that encodes cH9/1, then wereboosted with soluble cH6/1 protein, followed by cH5/1 protein (H5 head,H1 stalk), and finally challenged with a panel of H1N1 viruses (FIGS.30A-30F). Following infection with FM1 (FIGS. 30A and 30B),A/Netherlands/602/2009 (pH1N1) (FIGS. 30C and 30D) and PR8 viruses(FIGS. 30E and 30F), all cHA-vaccinated animals were protected fromchallenge and displayed only minimal amounts of weight loss, if any. Incontrast, negative control animals that received BSA following primingwith cH9/1 DNA lost considerable amounts of weight and, with theexception of one animal, succumbed to infection by day 9 (FIGS.30A-30F). The survival of the cHA-vaccinated animals in each of thechallenge experiments was significantly different from that of controls(FIGS. 30B, 30D and 30F). To confirm that the protection elicited was aresult of stalk-specific humoral immunity, all mice were confirmed to beHI negative to each challenge virus though the sera were capable ofbinding H1 HA by ELISA (FIG. 30G), confirming the production of stalkspecific antibodies by our vaccination protocol. Because it is possiblethat CD8 T cells directed towards epitopes within the HA stalk could beplaying a role in the protection seen here (see, e.g., M. Tamura et al.,J. Virol. 72:9404-9406), mice were vaccinated and depleted of CD8 Tcells by administering monoclonal antibody 2.43 prior to PR8 challenge(M. L. Salem, 2000, Int. J. Immunopharmacol. 22:707-718). Depletion didnot affect weight loss nor survival outcomes, implicating a humoralresponse in the protection elicited by vaccination (FIGS. 30H and 30I).Therefore, an adaptive humoral immune response towards the HA stalk, andnot the head, was providing protection against the three different H1N1viruses.

In order to further validate that the cHA-based vaccination protocolinduced stalk-specific antibodies with neutralizing capability againstother subtypes, the ability of purified IgG from vaccinated mice toblock the entry of pseudoparticles that harbor an H2 HA was tested.Because pseudoparticles express a luciferase reporter gene followingentry, neutralizing activity was measured by the absence of luciferaseenzymatic activity in cell supernatants (R. Hai et al., 2012, J. Virol.86:5774-5781 and N. Pica et al., 2012, PNAS 109:2573-2578). Consistentwith the protection seen following challenge, IgG purified fromvaccinated mice inhibited the entry of pseudoparticles in adose-dependent manner and with similar efficacy to that of CR6261, amonoclonal antibody with specificity to the HA stalk that was used asthe positive control (FIG. 30J). The vaccination protocol, therefore,elicited stalk antibodies with broad specificities, capable ofneutralizing other group 1 HAs like H2.

6.6.3 Conclusion

This example demonstrates the protective effect of a stalk-specificimmune response that can be elicited through vaccination with chimericHAs. It was demonstrated that an immune response directed towards the HAstalk was sufficient for protection from viral challenge, and that thisvaccination protocol provided heterosubtypic protection. A similarstrategy could be developed in humans to provide protection against abroad range of influenza viruses, negating the need for annualvaccination, and enhancing pandemic preparedness.

6.7 Example 7 A Carboxy-Terminal Trimerization Domain StabilizesConformational Epitopes on the Stalk Domain of Soluble RecombinantHemagglutinin Substrates

This example demonstrates that a carboxy-terminal trimerization domainis important to the structural integrity of stalk epitopes onrecombinant soluble influenza virus hemagglutinin.

6.7.1 Materials and Methods 6.7.1.1 Cells

Sf9 insect cells (ATCC # CRL-1711) were grown in TMN-FH medium (GeminiBio-Products) supplemented with 10% FBS (Atlanta Biologicals), 0.1%Pluronic F68 (Sigma) and a Penicillin-Streptomycin antibiotic (Gibco)mixture. BTI-TN-5B1-4 cells (High Five—Vienna Institute of Biotechnologysubclone) were grown in HyClone SFX serum free medium (FisherScientific) supplemented with Penicillin-Streptomycin antibiotic mixture(Gibco).

6.7.1.2 Cloning and Recombinant Baculovirus Generation

Sequences coding for HAs of H1 strains A/Puerto Rico/8/34 (PR8),A/California/04/09 (Cal09), H2 strain A/Japan/305/57 (JAP57), H3 strainsA/Hong Kong/1/68 (HK68), A/Wisconsin/67/05 (Wisc05) and H5 strain A/VietNam/1203/04 (VN04—with removed polybasic cleavage site; see Steel etal., 2009, J Virol 83: 1742-1753) were amplified from pCAGGS plasmids bypolymerase chain reaction and cloned into a modified pFastBac vector(Invitrogen) using BamHI or StuI and NotI restriction endonucleases(NEB). Two sets of constructs, HA without and with trimerization domain,were cloned: HA constructs without trimerization domain were designed sothat the C-terminal transmembranee- and endodomain of the HA werereplaced with a hexahistidine-tag (HA sequence ends with 1509 for H1,V509 for H2 and H5 and G508 for H3; H3 numbering); the other set ofconstructs, HA with a trimerization domain, also lack the C-terminaltransmembrane- and endodomains (HA sequence ends with V503—H3 numbering)but include a thrombin cleavage site and a T4 foldon trimerizationdomain (see, e.g., Meier et al., 2004, J Mol Biol 344: 1051-1069) inaddition to the C-terminal hexahistidine-tag (FIG. 31). Generatedrecombinant pFastBac clones were transformed into DH10Bac bacteria(Invitrogen) according to the manufacturer's instructions andrecombinant bacmids were prepared with a PureLink Plasmid FilterMidiprep kit (Invitrogen). Recombinant bacmids were transformed into Sf9cells using Cellfectin II (Invitrogen) for rescue of recombinantbaculovirus. All sequences were confirmed by Sanger sequencing.

6.7.1.3 Protein Expression, Purification and Characterization

Baculovirus was amplified in Sf9 cells to a passage 3 stock and thenused to infect BTI-TN-5B1-4 (High Five) cells at 1×10⁶ cells/ml inHyClone SFX serum free media (Fisher Scientific) at a multiplicity ofinfection of 10. Expression was carried out in 1000 ml shaker flasks for96 hours at 28° C. After 96 hours, supernatants were cleared by lowspeed centrifugation (5000 g, 4° C., 20 min) and incubated with Ni-NTA(Qiagen) resin (3 ml slurry for 250 ml of culture supernatant) for twohours at room temperature (RT). The resin-supernatant mixture was thenpassed over 10 ml polypropylene columns (Qiagen). The retained resin waswashed four times with 15 ml of washing buffer (50 mM Na₂HCO₃, 300 mMNaCl, 20 mM imidazole, pH 8) and protein was eluted with elution buffer(50 mM Na₂HCO₃, 300 mM NaCl, 300 mM imidazole, pH 8). The eluate wasconcentrated using Amicon Ultracell (Millipore) centrifugation unitswith a cut-off of 30 kDa and buffer was changed to phosphate bufferedsaline (PBS) of pH 7.4. Protein concentration was quantified usingQuickstart Bradford Dye Reagent (Bio-Rad) with a bovine serum albuminstandard curve. Protein purity, integrity and identity was assessed bysodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)(4-20% polyacrylamide—Mini PROTEAN TGX gels, Bio-Rad), Coomassiestaining and Western blot or enzyme linked immunosorbent assay (ELISA).Extent of trimerization and/or multimerization was tested bycrosslinking of HA with bis-[sulfosuccinimidyl]suberate (BS³—FisherScientific) according to the manufacturer's recommendations. Briefly, 3μg of HA were incubated in 30 μl of PBS in the presence of a 25 foldmolar excess of BS³ crosslinker. The mixture was incubated at RT for 30minutes and then BS³ was quenched by adding 1M Tris-HCl buffer (pH 8) toa final concentration of 50 mM. Subsequently SDS-PAGE and/or Westernblot analysis with a mouse anti-his primary antibody (Sigma) andanti-mouse horseradish peroxidase (Santa Cruz Biotechnology) or alkalinephosphatase (Santa Cruz Biotechnology) conjugated secondary antibody wasperformed.

6.7.1.4 Enzyme Linked Immunosorbent Assay

Immunolon 4HBX (Fisher Scientific) plates were coated with recombinantHA with and without trimerization domain at a concentration of 5 μg/mlin coating buffer (0.1 M Na2CO3/NaHCO3, pH 9.2, 50 μl/well) overnight at4° C. The plates were then blocked for one hour at RT with PBS (pH 7.4)containing 1% Tween 20 (TBPS) and 3% non-fat dry milk powder. Afterblocking, plates were washed once with TPBS and then incubated withthree fold dilutions of monoclonal antibody or sera (100 μl per well inTPBS with 1% milk powder—monoclonal antibody starting concentration 30μg/ml; 1:100 dilution for sera) for one hour at RT. Plates were thenwashed trice with 100 μl of TPBS and incubated for another hour at RTwith horse radish peroxidase conjugated anti-mouse IgG (Santa CruzBiotechnology) or anti-human Fab secondary antibody (Sigma) at adilution of 1:3000 (50 μl per well). After three more washes, plateswere developed using SigmaFAST OPD substrate (Sigma) (100 μl/well),stopped with 3M HCl (50 μl/well) and read at an absorption of 490 nm ona Synergy 4 (BioTek) plate reader. The obtained read-out was backgroundsubtracted with values from secondary antibody-only incubated wells.

For stability studies, HA from PR8 virus with trimerization domain wasstored at 4° C. for 60 days, or at −80° C. and went through one(standard), two, three or four freeze-thaw cycles. Stability of headversus stalk binding antibodies was compared using PY102 and C179monoclonal antibodies. Antibody-HA combinations in ELISA were done intriplicates except for stability studies where duplicates were used.

6.7.2 Results 6.7.2.1 A C-Terminal Trimerization Domain Stabilizes HAsand Induces Trimer Formation

The extracellular domain of various group 1 and group 2 HAs wereexpressed in soluble form with or without a C-terminal T4 phagetrimerization domain (FIG. 31) in the baculoviral expression system.Proteins were harvested 96 hours post infection and purified via aC-terminal hexahistidine-tag using a Ni-NTA column. Purified protein wasconcentrated using ultrafiltration spin columns, assessed for proteinintegrity and impurities by SDS-PAGE and Coomassie staining andquantified with Bradford reagent. Based on the amino acid sequence andthe fact that baculovirus expressed full length HAs without polybasiccleavage site are usually uncleaved, the extracellular domain of HAwould have an expected molecular mass of approximately 60 kDa permonomer (or 180 kDa per trimer) without taking glycosylation intoaccount. Cal09 (H1), JAP57 (H2) and VN04 (H5 without polybasic cleavagesite) HA without trimerization domain seemed to be partially cleavedinto HA1 and HA2 as indicated by the presence of bands at approximately40 kDa (HA1) and 25 kDa (HA2) in addition to the uncleaved HA band at 60kDa (HA0). Based on the exclusive presence of a 60 kDa band for Cal09,JAP57 and VN04 HAs with trimerization domains in the non-reducing,denaturing SDS-PAGE, it can be assumed that these proteins are expressedmostly as an uncleaved HA0 (FIG. 32A). Additionally, preparations ofWisc05 (H3) HA without a trimerization domain showed a degradationproduct at 40 kDa that was reactive when probed with an anti-stalkantibody (12D1). This species was thus likely a product of non-specificcleavage. Wisc05 HA with trimerization domain appeared only as an HA0band (FIG. 32A). PR8 and HK68 HA appeared to be very stable (present asHA0) even in the absence of a trimerization domain.

HAs were crosslinked with and without T4 trimerization domain using BS3,a hydrophilic 11 Angstrom chemical crosslinker that was recently used toshow trimerization for HAs (see, e.g., Weldon et al., 2010, PLoS One 5).After crosslinking, samples were diluted in a reducing, denaturingloading dye and resolved on a reducing, denaturing SDS-PAGE gel. Group 1HAs without trimerization domain formed high molecular weight oligomersthat barely ran into the running gel and were mostly retained in thestacking gel (FIGS. 32B and 32C). The strongest phenotype was detectedfor VN04 and JAP57; other group 1 HAs also formed additional trimers(approximately 230 kDa), dimers (130 to 150 kDa) and monomers (60 kDa)(FIGS. 32B and 32C). Group 1 HAs with trimerization domain formed mostlytrimers that ran at approximately 230 kD on the SDS-PAGE gel and formeda defined band in the running gel. However, they also formed dimers(approximately 130 to 150 kDa, strongest for Cal09) and monomers (60kDa). Group 2 HAs behaved differently: HK68 HA formed predominantlytrimers and to some degree dimers regardless of the presence of atrimerization domain. Wisc05 HA showed mainly dimerization in theabsence of a trimerization domain, while HA with the T4 domain wasmostly trimerized.

6.7.2.2 A C-Terminal Trimerization Domain Strongly Enhances Binding ofStalk-Reactive Antibodies to HA Substrates

The reactivity of a panel of broadly reactive, neutralizing antibodiesto the HA constructs was assessed in order to determine differentialbinding of these antibodies to HA substrates with and withouttrimerization domain. Stalk-specific antibodies mAb C179, mouse mAb6F12, human mAb CR6261 (all group 1 specific); and mouse mAb 12D1 andhuman mAb CR8020 (both group 2 specific) were used in the experiment.Four other stalk-reactive antibodies, KB2, BD3, GG3 and IB11, that wererecently isolated and characterized to have reactivity to both H1 and H5Has also were used in the experiment. As a control, strain specificantibodies that are known to bind to the globular head domain of HA wereused. As additional controls, sera of mice sub-lethally infected withinfluenza virus strains (PR8, Cal09, H3, VN04) or vaccinated with VLPs(JAP57) was used. Antibodies C179, CR6261 and 6F12 showed a strongbinding phenotype to both H1 HAs that were tested (Cal09 and PR8). It isof note that they bound exclusively to HAs that had a trimerizationdomain (FIGS. 33A and 33B); no binding was observed to HAs without atrimerization domain. Similar binding characteristics were seen with thefour other stalk-reactive broadly neutralizing H1-H5 antibodies. Incontrast, head-specific antibodies, such as 7B2 (Cal09) and PY102 (PR8),reacted with HAs irrespective of the expression of a trimerizationdomain and these findings were confirmed using sera from Cal09 or PR8infected animals (FIGS. 33A and 33B).

This effect is not specific to the H1 subtype—when testing the bindingof C179 and CR6261 to JAP57 (H2) and VN04 (H5) HAs with and without atrimerization domain, a similar phenotype was observed, where theseantibodies only reacted with trimerized forms of the protein (FIGS. 34Aand 34B). The same result was seen when reactivity of the four H1-H5antibodies was assessed. Head-specific antibodies 8F8 (JAP57) and mAb#8(VN04) or polyclonal anti-H2 or anti-H5 sera recognized both forms of HAequally well.

For group 2 HA-binding antibodies a different pattern emerged. In orderto test the effects of a trimerization domain on reactivity of stalkantibodies with group 2 HAs, broadly reactive antibodies CR8020 and 12D1were used. CR8020 binds a conformational epitope in group 2 HAs, while12D1 is thought to bind to a linear epitope within the long alpha helix(LAH) of the HA2 subunit. CR8020 binding to HK68 and Wisc05 HAs withtrimerization domains was greatly enhanced over binding to HAs withouttrimerization domain (FIGS. 35A and 35B). However, lack of thetrimerization domain did not completely abolish binding as seen withgroup 1 HAs. 12D1 did not distinguish between HAs with or without thetrimerization domain (FIG. 35).

6.7.3 Conclusion

The T4 trimerization domain allows for successful trimerization ofsoluble HA molecules and greatly increases the stability of thesemolecules following baculovirus expression.

6.8 Example 8 Influenza Virus Expressing a Chimeric Ha Comprising theStem Domain of an H1 Influenza Virus and the Globular Head Domain of anH5 Influenza Virus (cH5/1)

This example demonstrates that the engineering and rescue of aninfluenza virus expressing a chimeric HA comprising the stem domain ofan H1 influenza virus and the globular head domain of an H5 influenzavirus (i.e, an influenza virus comprising a genome engineered to expressa cH5/1 chimeric influenza hemagglutinin polypeptide).

A plasmid for rescue of influenza virus expressing a cH5/1 chimericinfluenza hemagglutinin polypeptide was constructed by substitutingcodons for the globular head domain (53 to 276, H3 numbering) in arescue plasmid that carries the HA gene of A/California/04/09 with theglobular head domain of A/Vietnam/1203/04 (H5). The sequence of theconstruct was confirmed by Sanger sequencing and expression in 293Tcells was tested using Western blot analysis in accordance with themethods described above.

Generation of recombinant virus for rescue was accomplished using theapproaches described in Example 5, above. The plasmid encoding thecH5/1_(Cal09) HA was co-transfected into 293T cells with 7 complementaryrescue plasmids that encode for the 7 other genomic segments of theinfluenza A virus (PR8 backbone). Supernatants were harvested on day onepost transfection and were directly inoculated into 10 day oldembryonated eggs. 48 hours post-inoculation eggs were chilled to 4° C.and allantoic fluid was harvested. Virus growth was assessed byhemagglutination assay. Supernatants from positive eggs were plagued onMDCK cells and single plaques were picked and propagated again inembryonated eggs. RNA from plaque purified viruses was isolated,reverse-transcribed and the sequence was confirmed by Sanger sequencing.The identity of the virus clones was proven by staining with a strictlyH1 stalk-reactive antibody (6F12) and a strain specific anti-headantibody against A/Vietnam/1203/04 (H5), confirming the generation ofand isolation of influenza virus comprising a genome engineered toexpress a cH5/1 chimeric influenza hemagglutinin polypeptide comprisingthe HA gene of A/California/04/09 and the globular head domain ofA/Vietnam/1203/04 (H5).

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

1. A chimeric influenza virus hemagglutinin (HA) polypeptide comprising:a. the stem domain of the HA from influenza virus A/California/4/2009(H1N1) and the globular head domain of the HA from an influenza virus ofthe H5 subtype; b. the stem domain of the HA from influenza virusA/Victoria/361/2011 (H3N2), A/harbor seal/Massachusetts/1/2011 (H3N8),or A/Indiana/10/2011 (H3N2) and the globular head domain of the HA froman influenza virus of the H5 subtype; c. the stem domain of the HA frominfluenza virus A/Victoria/361/2011 (H3N2), A/harborseal/Massachusetts/1/2011 (H3N8), or A/Indiana/10/2011 (H3N2) and theglobular head domain of the HA from an influenza virus of the H7subtype; d. the stem domain of the HA from influenza virusB/Malaysia/2506/2004, B/Florida/4/2006, B/Wisconsin/1/2010, orB/Brisbane/60/2008 and the globular head domain of the HA from aninfluenza virus of the H5 subtype; e. the stem domain of the HA frominfluenza virus B/Malaysia/2506/2004, B/Florida/4/2006,B/Wisconsin/1/2010, or B/Brisbane/60/2008 and the globular head domainof the HA from an influenza virus of the H7 subtype; or f. the stemdomain of the HA from influenza virus B/Malaysia/2506/2004,B/Florida/4/2006, B/Wisconsin/1/2010, or B/Brisbane/60/2008 and theglobular head domain of the HA from a different strain of influenza Bvirus.
 2. The chimeric influenza virus hemagglutinin (HA) polypeptide ofclaim 1(a), wherein the H5 subtype is influenza virusA/Vietnam/1203/2004 (H5), A/Indonesia/5/2005 (H5), A/Anhui/1/2005 (H5),A/Bar headed goose/Quinghai/1A/2005 (H5), A/turkey/Turkey/1/2005 (H5),or A/Whooperswan/Mongolia/244/2005 (H5).
 3. (canceled)
 4. The chimericinfluenza virus hemagglutinin (HA) polypeptide of claim 1(b), whereinthe H5 subtype is influenza virus HA of A/Vietnam/1203/2004 (H5),A/Indonesia/5/2005 (H5), A/Anhui/1/2005 (H5), A/Bar headedgoose/Quinghai/1A/2005 (H5), A/turkey/Turkey/1/2005 (H5), orA/Whooperswan/Mongolia/244/2005 (H5).
 5. (canceled)
 6. The chimericinfluenza virus hemagglutinin (HA) polypeptide of claim 1(c), whereinthe H7 subtype is influenza virus A/Netherlands/219/03 (H7),A/Canada/504/04 (H7), A/Canada/444/04 (H7), A/chicken/Jalisco/CPA1/2012(H7), A/mallard/Alberta/24/2001 (H7), A/Rhea/NC/39482/93 (H7), orA/mallard/Netherlands/12/2000 (H7).
 7. (canceled)
 8. The chimericinfluenza virus hemagglutinin (HA) polypeptide of claim 1(d), whereinthe H5 subtype is influenza virus A/Vietnam/1203/2004 (H5),A/Indonesia/5/2005 (H5), A/Anhui/1/2005 (H5), A/Bar headedgoose/Quinghai/1A/2005 (H5), A/turkey/Turkey/1/2005 (H5), orA/Whooperswan/Mongolia/244/2005 (H5).
 9. (canceled)
 10. The chimericinfluenza virus hemagglutinin (HA) polypeptide of claim 1(e), whereinthe H7 subtype is influenza virus A/Netherlands/219/03 (H7),A/Canada/504/04 (H7), A/Canada/444/04 (H7), A/chicken/Jalisco/CPA1/2012(H7), A/mallard/Alberta/24/2001 (H7), A/Rhea/NC/39482/93 (H7), orA/mallard/Netherlands/12/2000 (H7).
 11. (canceled)
 12. The chimericinfluenza virus hemagglutinin (HA) polypeptide of claim 1(f), whereinthe different strain of influenza B virus is B/Lee/1940 orB/seal/Netherlands/1/99.
 13. A nucleic acid encoding the chimericinfluenza virus hemagglutinin (HA) polypeptide of claim
 1. 14. A cellexpressing the nucleic acid of claim
 13. 15. A virus comprising a genomeengineered to express the nucleic acid of claim
 13. 16. A virus orvirus-like particle comprising the polypeptide of claim
 1. 17.(canceled)
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. (canceled)22. An immunogenic composition comprising the polypeptide of claim 1.23. (canceled)
 24. (canceled)
 25. (canceled)
 26. (canceled) 27.(canceled)
 28. An immunogenic composition comprising: A (i) a firstchimeric influenza virus hemagglutinin (HA) polypeptide, wherein thepolypeptide comprises the stem domain of the HA of influenza virusA/California/4/2009 (H1N1) and the globular head domain of the HA ofinfluenza virus /Vietnam/1203/2004 (H5), A/Indonesia/5/2005 (H5),A/Anhui/1/2005 (H5), A/Bar headed goose/Quinghai/1A/2005 (H5),A/turkey/Turkey/1/2005 (H5), or A/Whooperswan/Mongolia/244/2005 (H5);and (ii) a second chimeric influenza virus hemagglutinin (HA)polypeptide, wherein the polypeptide comprises (a) the stem domain ofthe HA of influenza virus A/Victoria/361/2011 (H3N2), A/harborseal/Massachusetts/1/2011 (H3N8), or A/Indiana/10/2011 (H3N2); and (b)the globular head domain of the HA of influenza virusA/Vietnam/1203/2004 (H5), A/Indonesia/5/2005 (H5), A/Anhui/1/2005 (H5),A/Bar headed goose/Quinghai/1A/2005 (H5), A/turkey/Turkey/1/2005 (H5),or A/Whooperswan/Mongolia/244/2005 (H5); or B (i) a first chimericinfluenza virus hemagglutinin (HA) polypeptide, wherein the polypeptidecomprises the stem domain of the HA of influenza virusA/California/4/2009 (H1N1) and the globular head domain of the HA ofinfluenza virus A/Vietnam/1203/2004 (H5), A/Indonesia/5/2005 (H5),A/Anhui/1/2005 (H5), A/Bar headed goose/Quinghai/1A/2005 (H5),A/turkey/Turkey/1/2005 (H5), or A/Whooperswan/Mongolia/244/2005 (H5);and (ii) a second chimeric influenza virus hemagglutinin (HA)polypeptide, wherein the polypeptide comprises (a) the stem domain ofthe HA of influenza virus A/Victoria/361/2011 (H3N2), A/harborseal/Massachusetts/1/2011 (H3N8), or A/Indiana/10/2011 (H3N2); and (b)the globular head domain of the HA of influenza virusB/Malaysia/2506/2004 and the globular head domain of the HA of influenzavirus A/Netherlands/219/03 (H7), A/Canada/504/04 (H7), A/Canada/444/04(H7), A/chicken/Jalisco/CPA1/2012 (H7), A/mallard/Alberta/24/2001 (H7),A/Rhea/NC/39482/93 (H7), or A/mallard/Netherlands/12/2000 (H7). 29.(canceled)
 30. The immunogenic composition of claim 28, furthercomprising a third chimeric influenza virus hemagglutinin (HA)polypeptide, wherein the polypeptide comprises (a) the stem domain ofthe HA of influenza virus B/Malaysia/2506/2004, B/Florida/4/2006,B/Wisconsin/1/2010; or B/Brisbane/60/2008; and (b) the globular headdomain of the HA of influenza virus A/Vietnam/1203/2004 (H5),A/Indonesia/5/2005 (H5), A/Anhui/1/2005 (H5), A/Bar headedgoose/Quinghai/1A/2005 (H5), A/turkey/Turkey/1/2005 (H5),A/Whooperswan/Mongolia/244/2005 (H5), B/Lee/1940, orB/Seal/Netherlands/1/99.
 31. An immunogenic composition comprising: A(i) a first virus comprising a genome engineered to express a firstnucleic acid that encodes a chimeric influenza virus hemagglutinin (HA)polypeptide, wherein the polypeptide comprises the stem domain of the HAof influenza virus A/California/4/2009 (H1N1) and the globular headdomain of the HA of influenza virus A/Vietnam/1203/2004 (H5),A/Indonesia/5/2005 (H5), A/Anhui/1/2005 (H5), A/Bar headedgoose/Quinghai/1A/2005 (H5), A/turkey/Turkey/1/2005 (H5), orA/Whooperswan/Mongolia/244/2005 (H5); and (ii) a second virus comprisinga genome engineered to express a second nucleic acid that encodes achimeric influenza virus hemagglutinin (HA) polypeptide, wherein thepolypeptide comprises (a) the stem domain of the HA of influenza virusA/Victoria/361/2011 (H3N2), A/harbor seal/Massachusetts/1/2011 (H3N8),or A/Indiana/10/2011 (H3N2); and (b) the globular head domain of the HAof influenza virus A/Vietnam/1203/2004 (H5), A/Indonesia/5/2005 (H5),A/Anhui/1/2005 (H5), A/Bar headed goose/Quinghai/1A/2005 (H5),A/turkey/Turkey/1/2005 (H5), or A/Whooperswan/Mongolia/244/2005 (H5); orB (i) a first virus comprising a genome engineered to express a firstnucleic acid that encodes a chimeric influenza virus hemagglutinin (HA)polypeptide, wherein the polypeptide comprises the stem domain of the HAof influenza virus A/California/4/2009 (H1N1) and the globular headdomain of the HA of influenza virus A/Vietnam/1203/2004 (H5),A/Indonesia/5/2005 (H5), A/Anhui/1/2005 (H5), A/Bar headedgoose/Quinghai/1A/2005 (H5), A/turkey/Turkey/1/2005 (H5), orA/Whooperswan/Mongolia/244/2005 (H5); and (ii) a second virus comprisinga genome engineered to express a second nucleic acid that encodes achimeric influenza virus hemagglutinin (HA) polypeptide, wherein thepolypeptide comprises (a) the stem domain of the HA of influenza virusA/Victoria/361/2011 (H3N2), A/harbor seal/Massachusetts/1/2011 (H3N8),or A/Indiana/10/2011 (H3N2); and (b) the globular head domain of the HAof influenza virus B/Malaysia/2506/2004 and the globular head domain ofthe HA of A/Netherlands/219/03 (H7), A/Canada/504/04 (H7),A/Canada/444/04 (H7), A/chicken/Jalisco/CPA1/2012 (H7),A/mallard/Alberta/24/2001 (H7), A/Rhea/NC/39482/93 (H7), orA/mallard/Netherlands/12/2000 (H7).
 32. (canceled)
 33. The immunogeniccomposition of claim 31, further comprising a third virus comprising agenome engineered to express a third nucleic acid that encodes achimeric influenza virus hemagglutinin (HA) polypeptide, wherein thepolypeptide comprises (a) the stem domain of the HA of influenza virusB/Malaysia/2506/2004, B/Florida/4/2006, B/Wisconsin/1/2010; orB/Brisbane/60/2008; and (b) the globular head domain of the HA ofinfluenza virus A/Vietnam/1203/2004 (H5), A/Indonesia/5/2005 (H5),A/Anhui/1/2005 (H5), A/Bar headed goose/Quinghai/1A/2005 (H5),A/turkey/Turkey/1/2005 (H5), A/Whooperswan/Mongolia/244/2005 (H5),B/Lee/1940, or B/Seal/Netherlands/1/99.
 34. An immunogenic compositioncomprising: A (i) a first virus comprising a first chimeric influenzavirus hemagglutinin (HA) polypeptide, wherein the polypeptide comprisesthe stem domain of the HA of influenza virus A/California/4/2009 (H1N1)and the globular head domain of the HA of influenza virusA/Vietnam/1203/2004 (H5), A/Indonesia/5/2005 (H5), A/Anhui/1/2005 (H5),A/Bar headed goose/Quinghai/1A/2005 (H5), A/turkey/Turkey/1/2005 (H5),or A/Whooperswan/Mongolia/244/2005 (H5); and (ii) a second viruscomprising a second chimeric influenza virus hemagglutinin (HA)polypeptide, wherein the polypeptide comprises (a) the stem domain ofthe HA of influenza virus A/Victoria/361/2011 (H3N2), A/harborseal/Massachusetts/1/2011 (H3N8), or A/Indiana/10/2011 (H3N2); and (b)the globular head domain of the HA of influenza virusA/Vietnam/1203/2004 (H5), A/Indonesia/5/2005 (H5), A/Anhui/1/2005 (H5),A/Bar headed goose/Quinghai/1A/2005 (H5), A/turkey/Turkey/1/2005 (H5),or A/Whooperswan/Mongolia/244/2005 (H5); or B (i) a first viruscomprising a first chimeric influenza virus hemagglutinin (HA)polypeptide, wherein the polypeptide comprises the stem domain of the HAof influenza virus A/California/4/2009 (H1N1) and the globular headdomain of the HA of influenza virus A/Vietnam/1203/2004 (H5),A/Indonesia/5/2005 (H5), A/Anhui/1/2005 (H5), A/Bar headedgoose/Quinghai/1A/2005 (H5), A/turkey/Turkey/1/2005 (H5), orA/Whooperswan/Mongolia/244/2005 (H5); and (ii) a second virus comprisinga second chimeric influenza virus hemagglutinin (HA) polypeptide,wherein the polypeptide comprises (a) the stem domain of the HA ofinfluenza virus A/Victoria/361/2011 (H3N2), A/harborseal/Massachusetts/1/2011 (H3N8), or A/Indiana/10/2011 (H3N2); and (b)the globular head domain of the HA of influenza virusA/Netherlands/219/03 (H7), A/Canada/504/04 (H7), A/Canada/444/04 (H7),A/chicken/Jalisco/CPA1/2012 (H7), A/mallard/Alberta/24/2001 (H7),A/Rhea/NC/39482/93 (H7), or A/mallard/Netherlands/12/2000 (H7). 35.(canceled)
 36. The immunogenic composition of claim 34, furthercomprising a third virus comprising a third chimeric influenza virushemagglutinin (HA) polypeptide, wherein the polypeptide comprises (a)the stem domain of the HA of influenza virus B/Malaysia/2506/2004,B/Florida/4/2006, B/Wisconsin/1/2010; or B/Brisbane/60/2008; and (b) theglobular head domain of the HA of influenza virus A/Vietnam/1203/2004(H5), A/Indonesia/5/2005 (H5), A/Anhui/1/2005 (H5), A/Bar headedgoose/Quinghai/1A/2005 (H5), A/turkey/Turkey/1/2005 (H5),A/Whooperswan/Mongolia/244/2005 (H5), B/Lee/1940, orB/Seal/Netherlands/1/99.
 37. (canceled)
 38. (canceled)
 39. (canceled)40. A method of immunizing a subject against influenza virus comprisingadministering to the subject an effective amount of the immunogeniccomposition of claim
 22. 41. A method of immunizing a subject againstinfluenza virus comprising administering to the subject a first dose ofan effective amount of an immunogenic composition and administering tothe subject a second dose of an effective amount of the immunogeniccomposition 30 days to 6 months after the subject has received the firstdose, wherein the immunogenic composition is the composition of claim22.
 42. (canceled)
 43. A method of immunizing a subject againstinfluenza virus comprising administering to the subject a first dose ofan effective amount of a first immunogenic composition and administeringto the subject a second dose of an effective amount of a secondimmunogenic composition 30 days to 6 months after the subject hasreceived the first dose, wherein the immunogenic compositions are thecompositions of claim 22, and wherein the globular head domain of thechimeric influenza virus hemagglutinin (HA) polypeptide present in thefirst immunogenic composition is different than the globular head domainof the chimeric influenza virus hemagglutinin (HA) polypeptide presentin the second immunogenic composition.
 44. A method of preventing aninfluenza virus disease or treating an influenza virus infection or aninfluenza virus disease comprising administering to a subject aneffective amount of the immunogenic composition of claim
 22. 45.(canceled)
 46. (canceled)
 47. (canceled)
 48. (canceled)
 49. (canceled)50. The chimeric influenza virus hemagglutinin (HA) polypeptide of claim1, wherein the polypeptide is soluble.
 51. (canceled)